Capsid-modified raav vectors and methods of use

ABSTRACT

Disclosed are tyrosine-modified rAAV vectors, as well as infectious virions, compositions, and pharmaceutical formulations that comprise them. Also disclosed are methods of preparing and methods for using the disclosed tyrosine-phosphorylated capsid protein mutant rAAV vectors in a variety of diagnostic and therapeutic applications including in vivo and ex vivo gene therapy, and large-scale production of rAAV vectors.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/680,668, filed Aug. 18, 2017, which is a continuation of U.S.application Ser. No. 14/847,528, filed Sep. 8, 2015, which is acontinuation of U.S. application Ser. No. 13/855,640, filed Apr. 2,2013, which is a continuation of U.S. application Ser. No. 12/595,196,filed Dec. 31, 2009, which is a national stage filing under 35 U.S.C. §371 of International PCT Application No. PCT/US2008/059647, filed Apr.8, 2008, which claims priority from provisional application Ser. No.60/910,798 filed Apr. 9, 2007, the entire contents of each of which areincorporated herein by reference in their entireties.

GOVERNMENT SPONSORSHIP

The invention was made with government support under Grant Nos.DK058327, HL051811, HL059412, HL078810, DK062302, EB002073, GM082946,HL065570, HL076901, and HL097088 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to the development of genedelivery vehicles. Also disclosed are improved rAAV vector compositionsuseful in expressing a variety of nucleic acid segments, including thoseencoding therapeutic proteins polypeptides, peptides, antisenseoligonucleotides, and ribozyme constructs, in various gene therapyregimens. Methods are also provided for preparing and using thesemodified rAAV-based vector constructs in a variety of viral-based genetherapies, and in particular, treatment and prevention of human diseasesusing conventional gene therapy approaches. The invention also providesrAAV-based vector delivery systems which may be used to assess therelative efficiency and infectivity of a variety of AAV particles havingmutations in one or more tyrosine residues of viral capsid proteins.

DESCRIPTION OF RELATED ART

Major advances in the field of gene therapy have been achieved by usingviruses to deliver therapeutic genetic material. The adeno-associatedvirus (AAV) has attracted considerable attention as a highly effectiveviral vector for gene therapy due to its low immunogenicity and abilityto effectively transduce non-dividing cells. AAV has been shown toinfect a variety of cell and tissue types, and significant progress hasbeen made over the last decade to adapt this viral system for use inhuman gene therapy.

In its normal “wild type” form, recombinant AAV (rAAV) DNA is packagedinto the viral capsid as a single stranded molecule about 4600nucleotides (nt) in length. Following infection of the cell by thevirus, the molecular machinery of the cell converts the single DNAstrand into a double-stranded form. Only the double-stranded DNA form isuseful to the polypeptides of the cell that transcribe the containedgene or genes into RNA.

AAV has many properties that favor its use as a gene deliveryvehicle: 1) the wild type virus is not associated with any pathologichuman condition; 2) the recombinant form does not contain native viralcoding sequences; and 3) persistent transgenic expression has beenobserved in many applications.

The transduction efficiency of recombinant adeno-associated virus 2(AAV) vectors varies greatly in different cells and tissues in vitro andin vivo. Systematic studies have been performed to elucidate thefundamental steps in the life cycle of AAV. For example, it has beendocumented that a cellular protein, FKBP52, phosphorylated at tyrosineresidues by epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK), inhibits AAV second-strand DNA synthesis and consequently,transgene expression in vitro^(24, 25) as well as in vivo.^(19, 27, 28)It has also been demonstrated that EGFR-PTK signaling modulates theubiquitin/proteasome pathway-mediated intracellular trafficking as wellas FKBP52-mediated second-strand DNA synthesis of AAV vectors. In thosestudies, inhibition of EGFR-PTK signaling led to decreasedubiquitination of AAV capsid proteins, which in turn, facilitatednuclear transport by limiting proteasome-mediated degradation of AAVvectors, implicating EGFR-PTK-mediated phosphorylation of tyrosineresidues on AAV capsids.

SUMMARY OF THE INVENTION

The present invention overcomes limitations and deficiencies inherent inthe prior art by providing novel rAAV-based genetic constructs thatencode one or more therapeutic agents useful in the preparation ofmedicaments for the prevention, treatment, and/or amelioration of one ormore diseases, disorders or dysfunctions resulting from a deficiency inone or more of such polypeptides. In particular, the invention providesAAV-based genetic constructs encoding one or more mammalian therapeuticagents (including, e.g., proteins, polypeptides, peptides, antibodies,antigen binding fragments, siRNAs, RNAis, antisense oligo- andpoly-nucleotides, ribozymes, and variants and/or active fragmentsthereof), for use in the diagnosis, prevention, treatment, and/oramelioration of symptoms of a variety of mammalian diseases, disorders,dysfunctions, trauma, injury, and such like.

Based on the inventors' latest discoveries, a new category of modifiedrAAV vectors have been developed which provide a higher-efficiencytransduction into selected cells than conventional and wild-type rAAVvectors.

By studying site-directed mutational analyses of surface-exposedtyrosine residues on various AAV capsid proteins, the inventors haveidentified that removal of one or more virion surface-presentingtyrosine residues (which provide a crucial signal for ubiquitination ofcapsid proteins) yield novel rAAV vectors and viral particles comprisingthem that bypass the ubiquitination step, thereby avoidingproteasome-mediated degradation, resulting in high-efficiencytransduction. The creation of these new vectors dramatically reduces thenumber of viral particles needed for conventional gene therapy regimens.The resulting tyrosine-modified AAV vectors described herein are moreefficient, more stable, less immunogenic, and produced at much lowercost than traditional vectors currently employed in human gene therapy.

Importantly, the methods of the present invention facilitate theproduction of novel AAV vectors with mutation of one or moresurface-exposed tyrosine residues on capsid proteins. These novelmutated vectors avoid degradation by the proteasome, and thussignificantly increase the transduction efficiency of these vectors. Theinventors have demonstrated that mutation of one or more of the tyrosineresidues on the outer surface of the capsid proteins [including, forexample, but not limited to, mutation of Tyr252 to Phe272 (Y252F),Tyr272 to Phe272 (Y272F), Tyr444 to Phe444 (Y444F), Tyr500 to Phe500(Y500F), Tyr700 to Phe700 (Y700F), Tyr704 to Phe704 (Y704F), and Tyr730to Phe730 (Y730F)] resulted in improved transduction efficiency of therAAV vectors when compared to wild-type.

In one aspect, the invention provides compositions comprisingrecombinant adeno-associated viral (AAV) vectors, virions, viralparticles, and pharmaceutical formulations thereof, useful in methodsfor delivering genetic material encoding one or more beneficial ortherapeutic product(s) to mammalian cells and tissues. In particular,the compositions and methods of the invention provide a significantadvancement in the art through their use in the treatment, prevention,and/or amelioration of symptoms of one or more mammalian diseases. It iscontemplated that human gene therapy will particularly benefit from thepresent teachings by providing new and improved viral vector constructsfor use in the treatment of a number of diverse diseases, disorders, anddysfunctions.

In another aspect, the invention concerns modified rAAV vector thatencode one or more mammalian therapeutic agents for the prevention,treatment, and/or amelioration of one or more disorders in the mammalinto which the vector construct is delivered. In particular, theinvention provides rAAV-based expression constructs that encode one ormore mammalian therapeutic agent(s) (including, but not limited to, forexample, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies,antigen binding fragments, as well as variants, and/or active fragmentsthereof, for use in the treatment, prophylaxis, and/or amelioration ofone or more symptoms of a mammalian disease, dysfunction, injury, and/ordisorder.

In another embodiment, the invention concerns genetically modified rAAVvectors that comprise at least a first nucleic acid segment that encodesone or more therapeutic agents that alter, inhibit, reduce, prevent,eliminate, or impair the activity of one or more endogenous biologicalprocesses in the cell. In particular embodiments, such therapeuticagents may be those that selectively inhibit or reduce the effects ofone or more metabolic processes, dysfunctions, disorders, or diseases.In certain embodiments, the defect may be caused by injury or trauma tothe mammal for which treatment is desired. In other embodiments, thedefect may be caused the over-expression of an endogenous biologicalcompound, while in other embodiments still; the defect may be caused bythe under-expression or even lack of one or more endogenous biologicalcompounds.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, siRNAs,and/or antisense oligonucleotides, to a particular cell transfected withthe vector, one may employ the modified AAV vectors disclosed herein byincorporating into the vector at least a first exogenous polynucleotideoperably positioned downstream and under the control of at least a firstheterologous promoter that expresses the polynucleotide in a cellcomprising the vector to produce the encoded therapeutic agent,including for example, peptides, proteins, polypeptides, antibodies,ribozymes, siRNAs, and antisense oligo- or polynucleotides. Suchconstructs may employ one or more heterologous promoters to express thetherapeutic agent of interest. Such promoters may be constitutive,inducible, or even cell- or tissue-specific. Exemplary promotersinclude, but are not limited to, a CMV promoter, a β-actin promoter, ahybrid CMV promoter, a hybrid β-actin promoter, an EF1 promoter, a U1apromoter, a U1b promoter, a Tet-inducible promoter, a VP16-LexApromoter, a joint-specific promoter and a human-specific promoter.

The genetically-modified rAAV vectors or expression systems of theinvention may also further comprise a second nucleic acid segment thatcomprises, consists essentially of, or consists of, one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise a second nucleic acid segment that comprises, consistsessentially of, or consists of, at least a first CMV enhancer, asynthetic enhancer, or a cell- or tissue-specific enhancer. The secondnucleic acid segment may also further comprise, consist essentially of,or consist of one or more intron sequences, post-transcriptionalregulatory elements, or such like. The vectors and expression systems ofthe invention may also optionally further comprise a third nucleic acidsegment that comprises, consists essentially of, or consists of, one ormore polylinker or multiple restriction sites/cloning region(s) tofacilitate insertion of one or more selected genetic elements,polynucleotides, and the like into the rAAV vectors at a convenientrestriction site.

In aspects of the invention, the exogenous polynucleotides that arecomprised within one or more of the improved rAAV vectors disclosedherein are preferably of mammalian origin, with polynucleotides encodingpolypeptides and peptides of human, primate, murine, porcine, bovine,ovine, feline, canine, equine, epine, caprine, or lupine origin beingparticularly preferred.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, enzymes, antibodies,siRNAs, ribozymes, or antisense polynucleotides, oligonucleotides, PNAmolecules, or a combination of two or more of these therapeutic agents.In fact, the exogenous polynucleotide may encode two or more suchmolecules, or a plurality of such molecules as may be desired. Whencombinational gene therapies are desired, two or more differentmolecules may be produced from a single rAAV expression system, oralternatively, a selected host cell may be transfected with two or moreunique rAAV expression systems, each of which may comprise one or moredistinct polynucleotides that encode a therapeutic agent.

In other embodiments, the invention also provides genetically-modifiedrAAV vectors that are comprised within an infectious adeno-associatedviral particle or a virion, or pluralities of such particles, whichthemselves may also be comprised within one or more diluents, buffers,physiological solutions or pharmaceutical vehicles, formulated foradministration to a mammal such as a human for therapeutic, and/orprophylactic gene therapy regimens. Such vectors, virus particles,virions, and pluralities thereof may also be provided in excipientformulations that are acceptable for veterinary administration toselected livestock, exotic or domesticated animals, companion animals(including pets and such like), as well as non-human primates,zoological or otherwise captive specimens, and such like, wherein theuse of such vectors and related gene therapy is indicated to produce abeneficial effect upon administration to such an animal.

The invention also concerns host cells that comprise at least one of thedisclosed rAAV vectors, virus particles, or virions. Such host cells areparticularly mammalian host cells, with human host cells beingparticularly highly preferred, and may be either isolated, in cell ortissue culture. In the case of genetically modified animal models, thetransformed host cells may even be comprised within the body of anon-human animal itself.

In certain embodiments, the creation of recombinant non-human hostcells, and/or isolated recombinant human host cells that comprise one ormore of the disclosed rAAV vectors is also contemplated to be useful fora variety of diagnostic, and laboratory protocols, including, forexample, means for the production of large-scale quantities of the rAAVvectors described herein. Such virus production methods are particularlycontemplated to be an improvement over existing methodologies includingin particular, those that require very high titers of the viral stocksin order to be useful as a gene therapy tool. The inventors contemplatethat one very significant advantage of the present methods will be theability to utilize lower titers of viral particles in mammaliantransduction protocols, yet still retain transfection rates at asuitable level.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, or host cells also formpart of the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in therapy, and for use in the manufacture of medicaments forthe treatment of one or more mammalian diseases, disorders,dysfunctions, or trauma. Such pharmaceutical compositions may optionallyfurther comprise one or more diluents, buffers, liposomes, a lipid, alipid complex; or the tyrosine-modified rAAV vectors may be comprisedwithin a microsphere or a nanoparticle. Pharmaceutical formulationssuitable for intramuscular, intravenous, or direct injection into anorgan or tissue or a plurality of cells or tissues of a human or othermammal are particularly preferred, however, the compositions disclosedherein may also find utility in administration to discreet areas of themammalian body, including for example, formulations that are suitablefor direct injection into one or more organs, tissues, or cell types inthe body. Such injection sites include, but are not limited to, thebrain, a joint or joint capsule, a synovium or subsynovium tissue,tendons, ligaments, cartilages, bone, peri-articular muscle or anarticular space of a mammalian joint, as well as direct administrationto an organ such as the heart, liver, lung, pancreas, intestine, brain,bladder, kidney, or other site within the patient's body, including, forexample, introduction of the viral vectors via intraabdominal,intrathorascic, intravascular, or intracerebroventricular delivery.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise,consist essentially of, or consist of, one or more of the rAAV vectorsdisclosed herein, such as for example pharmaceutical formulations of thevectors intended for administration to a mammal through suitable means,such as, by intramuscular, intravenous, intra-articular, or directinjection to one or more cells, tissues, or organs of a selected mammal.Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed rAAV vectors, virions,viral particles, transformed host cells or pharmaceutical compositionscomprising such; and instructions for using the kit in a therapeutic,diagnostic, or clinical embodiment also represent preferred aspects ofthe present disclosure. Such kits may further comprise one or morereagents, restriction enzymes, peptides, therapeutics, pharmaceuticalcompounds, or means for delivery of the composition(s) to host cells, orto an animal (e.g., syringes, injectables, and the like). Such kits maybe therapeutic kits for treating, preventing, or ameliorating thesymptoms of a disease, deficiency, dysfunction, and/or injury, and maycomprise one or more of the modified rAAV vector constructs, expressionsystems, virion particles, or a plurality of such particles, andinstructions for using the kit in a therapeutic and/or diagnosticmedical regimen. Such kits may also be used in large-scale productionmethodologies to produce large quantities of the viral vectorsthemselves (with or without a therapeutic agent encoded therein) forcommercial sale, or for use by others, including e.g., virologists,medical professionals, and the like.

Another important aspect of the present invention concerns methods ofuse of the disclosed rAAV vectors, virions, expression systems,compositions, and host cells described herein in the preparation ofmedicaments for preventing, treating or ameliorating the symptoms ofvarious diseases, dysfunctions, or deficiencies in an animal, such as avertebrate mammal. Such methods generally involve administration to amammal, or human in need thereof, one or more of the disclosed vectors,virions, viral particles, host cells, compositions, or pluralitiesthereof, in an amount and for a time sufficient to prevent, treat, orlessen the symptoms of such a disease, dysfunction, or deficiency in theaffected animal. The methods may also encompass prophylactic treatmentof animals suspected of having such conditions, or administration ofsuch compositions to those animals at risk for developing suchconditions either following diagnosis, or prior to the onset ofsymptoms.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, ribozymes, or antisenseoligonucleotides, or a combination of these. In fact, the exogenouspolynucleotide may encode two or more such molecules, or a plurality ofsuch molecules as may be desired. When combinational gene therapies aredesired, two or more different molecules may be produced from a singlerAAV expression system, or alternatively, a selected host cell may betransfected with two or more unique rAAV expression systems, each ofwhich will provide unique heterologous polynucleotides encoding at leasttwo different such molecules.

In other embodiment, the invention also concerns the disclosed rAAVvectors comprised within an infectious adeno-associated viral particle,comprised within one or more pharmaceutical vehicles, and may beformulated for administration to a mammal such as a human fortherapeutic, and/or prophylactic gene therapy regimens. Such vectors mayalso be provided in pharmaceutical formulations that are acceptable forveterinary administration to selected livestock, domesticated animals,pets, and the like.

The invention also concerns host cells that comprise the disclosed rAAVvectors and expression systems, particularly mammalian host cells, withhuman host cells being particularly preferred.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, host cells also form partof the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in the manufacture of medicaments and methods involvingtherapeutic administration of such rAAV vectors. Such pharmaceuticalcompositions may optionally further comprise liposomes, a lipid, a lipidcomplex; or the rAAV vectors may be comprised within a microsphere or ananoparticle. Pharmaceutical formulations suitable for intramuscular,intravenous, or direct injection into an organ or tissue of a human areparticularly preferred.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise oneor more of the AAV vectors disclosed herein, such as for examplepharmaceutical formulations of the vectors intended for administrationto a mammal through suitable means, such as, by intramuscular,intravenous, or direct injection to cells, tissues, or organs of aselected mammal. Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed vectors, virions, hostcells, viral particles or compositions; and (ii) instructions for usingthe kit in therapeutic, diagnostic, or clinical embodiments alsorepresent preferred aspects of the present disclosure. Such kits mayfurther comprise one or more reagents, restriction enzymes, peptides,therapeutics, pharmaceutical compounds, or means for delivery of thecompositions to host cells, or to an animal, such as syringes,injectables, and the like. Such kits may be therapeutic kits fortreating or ameliorating the symptoms of particular diseases, and willtypically comprise one or more of the modified AAV vector constructs,expression systems, virion particles, or therapeutic compositionsdescribed herein, and instructions for using the kit.

Another important aspect of the present invention concerns methods ofuse of the disclosed vectors, virions, expression systems, compositions,and host cells described herein in the preparation of medicaments fortreating or ameliorating the symptoms of various polypeptidedeficiencies in a mammal. Such methods generally involve administrationto a mammal, or human in need thereof, one or more of the disclosedvectors, virions, host cells, or compositions, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a deficiencyin the affected mammal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thefollowing description taken in conjunction with the accompanyingdrawings, in which like reference numerals identify like elements, andin which:

FIG. 1A and FIG. 1B show the AAV2-mediated transgene expression in HeLacells, pre-treated with or without Tyr23, following transduction witheither ssAAV2-EGFP or scAAV2-EGFP vectors. FIG. 1A shows transgeneexpression was detected by fluorescence microscopy at 48 hrpost-infection. Original magnification 100×. FIG. 1B shows quantitativeanalyses of AAV2 transduction efficiency. Images from five visual fieldswere analyzed quantitatively by ImageJ® analysis software. Transgeneexpression was assessed as total area of green fluorescence (pixel²) pervisual field (mean±SD). Analysis of variance (ANOVA) was used to comparetest results with the control and they were determined to bestatistically significant. *P<0.05 vs. control+ssAAV2-EGFP; # P<0.05 vs.control+scAAV2-EGFP.

FIG. 2A and FIG. 2B illustrate the AAV-mediated transgene expression inHeLa cells mock-transfected, or stably transfected with wt- or C-Smutant TC-PTP expression plasmids, following transduction with eitherssAAV2-EGFP or scAAV2-EGFP vectors. FIG. 2A shows transgene expressionwas detected by fluorescence microscopy at 48-hr post-infection(original magnification: 100×). FIG. 2B illustrates the quantitativeanalyses of AAV2 transduction efficiency was assessed as described inthe legend to FIG. 1A and FIG. 1B, and were determined to bestatistically significant. *P<0.05 vs. control+ssAAV2-EGFP; # P<0.05 vs.control+scAAV2-EGFP.

FIG. 3A and FIG. 3B show Southern blot analyses of cytoplasmic andnuclear distribution of AAV2 genomes in HeLa cells followingpre-treatment with Tyr23, over-expression of wtTC-PTP, or treatment withMG132 (FIG. 3A), and densitometric scanning of autoradiographs for thequantitation of relative amounts of viral genomes (FIG. 3B). Theseresults are representative of two independent studies.

FIG. 4A and FIG. 4B show the comparative analyses of AAV2 transductionefficiency in HeLa cells with various treatments. FIG. 4A shows HeLacells mock-treated or treated with Tyr23, MG132 or both, and cellsstably transfected with the wt TC-PTP expression plasmid were eithermock-treated or treated with MG132 followed by infection with AAV-lacZvectors. Cells were fixed and stained with X-Gal. Transgene expressionwas detected by microscopy at 48-hr post-infection (originalmagnification: 100×). FIG. 4B shows the quantitative analyses of AAVtransduction efficiency assessed as described in the legend to FIG. 1Aand FIG. 1B, and were determined to be statistically significant.*P<0.05 vs. control+ssAAV2-lacZ.

FIG. 5A and FIG. 5B show the comparative analyses of AAV2-mediatedtransduction efficiency in HeLa cells with various treatments, followingtransduction with scAAV2-EGFP vectors. FIG. 5A shows HeLa cellsmock-treated or treated with Tyr23, MG132, or both, and cells eithermock-transfected or stably transfected with the wt- or mTC-PTPexpression plasmids were either mock-treated or treated with MG132.Transgene expression was detected by fluorescence microscopy at 48 hrpost-infection (original magnification 100×). FIG. 5B: Quantitativeanalyses of AAV transduction efficiency was assessed as described in thelegend to FIG. 1A and FIG. 1B, and were determined to be statisticallysignificant. *P<0.05 vs. control+scAAV2-EGFP.

FIG. 6 shows the western blot analyses of ubiquitinated proteins in HeLacells following treatment with MG132 in the presence or absence of Tyr23or TC-PTP. Whole cell lysates (WCL) prepared from untreated cells (lanes1 and 6), and following treatment with MG132 (lanes 2 and 7), Tyr23(lane 3), or both (lane 8), and cells either stably transfected with thewt- or mTC-PTP expression plasmids following either mock-treatment(lanes 4 and 5) or treatment with MG132 (lanes 9 and 10) were probedwith anti-Ub monoclonal antibody.

FIG. 7 shows the western blot analyses of ubiquitinated AAV2 capsidproteins in HeLa cells treated with MG132 in the presence or absence ofTyr23 or wtTC-PTP, following transduction with ssAAV2-RFP vectors. WCLprepared from HeLa cells untreated or treated with MG132 followingmock-infected (lane 1 and 2), and HeLa cells untreated (lane 3), treatedwith Tyr23 (lane 4), MG132 (lane 5), or both (lane 6), or cells stablytransfected with the wtTC-PTP expression plasmid following eithermock-treatment (lane 7) or treatment with MG132 (lane 8), followinginfection with ssAAV2-RFP vectors were immunoprecipitated with anti-AAV2capsid antibody A20 followed by Western blot analyses with anti-Ubmonoclonal antibody.

FIG. 8 shows a model for interaction between EGFR-PTK signaling andubiquitin/proteasome pathway in the regulation of intracellulartrafficking as well as second-strand DNA synthesis of AAV2 vectors.Early endosome (EE); clathrin-coated pits (CP); late endosome (LE);FKBP52 (F); phosphotyrosine residues (P).

FIG. 9 shows the tyrosine-dephosphorylation of FKBP52, either bypre-treatment with Tyr23 or over-expression of TC-PTP, does not affectGFP gene expression following plasmid-mediated transfection in HeLacells.

FIG. 10A and FIG. 10B show the transduction efficiency of neither ssAAV(FIG. 10A) nor rAAV vectors (FIG. 10B) in HeLa cells over-expressingTC-PTP, or following pre-treatment with Tyr23, was further enhanced bytreatment with MG132 under non-saturating conditions (1,000 or 2,000viral particles/cell) of transduction.

FIG. 11 shows the in vitro phosphorylation of AAV2 capsids by EGFR-PTKfrom two different packaging systems was analyzed by Western Blottingusing anti-p-Tyr antibody for detection of phosphotyrosine containingcapsid proteins. K9: AAV2-adiponectin (baculovirus-based heterologousrAAV packaging system); RFP: ssAAV2-RFP (293 cells-based rAAV packagingsystem); ds-EGFP: scAAV2-CB-EGFP (293 cells-based rAAV packagingsystem).

FIG. 12 shows the in vitro phosphorylation of AAV2 capsids by EGFR-PTKfollowed by separating intact virions and free capsid proteins usingcentrifugal filter devices [(Ultracel™ YM-100 (kDa) and YM-30 (kDa)] wasanalyzed by Western blotting using anti-p-Tyr antibody for detection ofphosphotyrosine containing capsid proteins and anti-AAV cap (B1)antibody for detection of total capsid proteins. K9: AAV2-adiponectin(baculovirus-based heterologous rAAV packaging system).

FIG. 13 shows the slot blot analysis for AAV2 entry to HeLa cells afterin vitro phosphorylation of AAV capsids by EGFR-PTK. HeLa cells wereinfected by AAV2-LacZ vectors, which were pre-incubated with ATP,EGFR-TPK or both. Low-M_(r) DNA samples were isolated at 2 hrpost-infection and analyzed by Slot blot hybridization using a³²P-labeled LacZ DNA probe.

FIG. 14A and FIG. 14B detail the comparative analyses of ssAAV2-mediatedtransduction efficiency in HeLa cells after in vitro phosphorylation ofAAV capsids by EGFR-PTK. FIG. 14A: HeLa cells were infected byssAAV2-RFP vectors, which were pre-incubated with ATP, EGFR-TPK or both.Transgene expression was detected by fluorescence microscopy at 48 hrpost-infection (original magnification: 100×). FIG. 14B shows thequantitative analyses of AAV2 transduction efficiency. Images from fivevisual fields were analyzed quantitatively by ImageJ® analysis software.Transgene expression was assessed as total area of red fluorescence(pixel²) per visual field (mean±SD). Analysis of variance (ANOVA) wasused to compare test results with the control and they were determinedto be statistically significant. *P<0.05 vs. ssAAV2-RFP.

FIG. 15A and FIG. 15B illustrate the comparative analyses ofscAAV2-mediated transduction efficiency in HeLa cells after in vitrophosphorylation of AAV2 capsids by EGFR-PTK. In FIG. 15A HeLa cells wereinfected by scAAV2-EGFP vectors, which were pre-incubated with ATP,EGFR-TPK or both. Transgene expression was detected by fluorescencemicroscopy at 48 hr post-infection (original magnification: 100×). FIG.15B shows the quantitative analyses of AAV2 transduction efficiencyassessed as described in the legend to FIG. 14A and FIG. 14B, anddetermined to be statistically significant. *P<0.05 vs. scAAV2-EGFP.

FIG. 16A and FIG. 16B depict Southern hybridization analyses ofcytoplasmic and nuclear distribution of AAV2 genomes in HeLa cells afterin vitro phosphorylation of AAV2 capsids by EGFR-PTK. HeLa cells wereinfected by AAV2-LacZ vectors, which were pre-incubated with ATP,EGFR-TPK or both. Low-M_(r) DNA samples were isolated at 18 hrpost-infection and electrophoresed on 1% agarose gels followed analyzedby Southern blot hybridization using a ³²P-labeled LacZ DNA probe (FIG.16A), and densitometric scanning of autoradiographs for the quantitationof relative amounts of viral genomes (FIG. 16B).

FIG. 17A and FIG. 17B show AAV2-mediated transduction of hepatocytesfrom normal C57BL/6 mice injected via tail vein with tyrosine-mutantcapsid scAAV2-EGFP vectors. FIG. 17A shows transgene expression wasdetected by fluorescence microscopy 2 weeks post-injection of 1×10¹⁰viral particles/animal via the tail vein (n=2 per experimental group)(original magnification: 50×). FIG. 17B illustrates quantitation of thetransduction efficiency in hepatocytes in C57BL/6 mice. *P<0.01 vs. WTscAAV2-EGFP.

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show comparative analyses ofAAV2-mediated transduction efficiency in HeLa C12 cells with or withoutco-infection with adenovirus, and treatment with proteasome- orEGFR-PTK-inhibitors following transduction with tyrosine-mutant capsidscAAV2-EGFP vectors. FIG. 18A shows cells mock-infected or infected withadenovirus, following transduction with the WT, Y444F or Y730F AAV2-EGFPvectors (original magnification: 100×). FIG. 18B illustratesquantitation of the transduction efficiency in HeLa C12 cells. Shown inFIG. 18C are cells that were mock-treated or treated with Tyr23 orMG132, following transduction with the WT or Y730F AAV2-EGFP vectors(original magnification: 100×). FIG. 18D illustrates quantitation of thetransduction efficiency. *P<0.05 vs. control.

FIG. 19 illustrates a western hybridization analysis of ubiquitinatedAAV2 capsid proteins in HeLa cells following transduction withtyrosine-mutant scAAV2-EGFP vectors. Whole cell lysates (WCL) preparedfrom cells, untreated or treated with MG132, following mock-infection(lanes 1 and 2), or infected with the WT (lanes 3 and 4), Y730F (lanes 5and 6), or Y444F (lanes 7 and 8) scAAV2-EGFP vectors wereimmunoprecipitated with anti-AAV2 capsid antibody A20 followed byWestern blot analyses with anti-Ub monoclonal antibody P4D1.

FIG. 20A and FIG. 20B depict Southern hybridization analyses forintracellular trafficking of the WT and tyrosine-mutant scAAV2-EGFPvectors and cytoplasmic [C] and nuclear [N] distribution of AAV2genomes. HeLa cells were mock-infected (lanes 1 and 2) or infected withthe WT (lanes 2 and 3), Y730F (lanes 5 and 6) or Y444F (lanes 7 and 8)scAAV2-EGFP vectors. In FIG. 20A, nuclear and cytoplasmic fractions wereobtained 18 hr post-infection, low-M_(r) DNA samples were isolated andelectrophoresed on 1% agarose gels followed by Southern blothybridization using a ³²P-labeled lacZ DNA probe. In FIG. 20Bquantitation of relative amounts of viral genomes is demonstrated. Theseresults are representative of two independent studies.

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D illustrate comparativeanalyses of the WT or Y730F ssAAV2-ApoE/hAAT-hF.IX vector-mediatedtransduction efficiency in hepatocytes in mice in vivo. Human F.IX(hF.IX) expression in plasma was determined as a function of time afterinjection of 1×10¹¹ viral particles/animal in BALB/c (FIG. 21A), andC3H/HeJ (FIG. 21B) mice via tail vein (tv), and 1×10¹⁰ viralparticles/animal in C57BL/6 mice via tail vein (tv) (FIG. 21C), orportal vein (pv) (FIG. 21D). Fold-increase of hF.IX peak levels of Y730Fvectors compared to the WT capsid vectors is indicated for each panel.Data are mean±SD (n=4 per experimental group).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would nevertheless be a routineundertaking for those of ordinary skill in the art having the benefit ofthis disclosure.

The adeno-associated virus 2 (AAV2) is a non-pathogenic human parvoviruswhich has gained attention as an alternative to the more commonly usedretrovirus- and adenovirus-based vectors for gene transfer and genetherapy. Recombinant AAV2 vectors have been shown to transduce a widevariety of cells and tissues in vitro and in vivo, and are currently inuse in Phase I/II clinical trials for gene therapy of a number ofdiseases such as cystic fibrosis, α-1 antitrypsin deficiency,Parkinson's disease, Batten's disease, and muscular dystrophy.Systematic studies have been undertaken to elucidate some of thefundamental steps in the life cycle of AAV2 vectors, which include viralbinding and entry, intracellular trafficking, uncoating, second-strandDNA synthesis and transgene expression, and viral genome integrationinto the host cell chromosome.

The ubiquitin-proteasome pathway has been shown to play an essentialrole in AAV2 intracellular trafficking. It has also been observed thatperturbations in EGFR-PTK signaling affects AAV2 transduction efficiencyby not only augmenting viral second-strand DNA synthesis, but also byfacilitating intracellular trafficking from the cytoplasm to thenucleus. Previously it was reported that intact AAV2 capsids could bephosphorylated at tyrosine residues by EGFR-PTK, but not atserine/threonine residues by casein kinase II (CKII) under cell-freeconditions in vitro, and that tyrosine-phosphorylation of AAV2 capsidsnegatively affects viral intracellular trafficking and transgeneexpression in intact cells in vivo. Based on these studies, it washypothesized that EGFR-PTK-mediated phosphorylation of capsid proteinsat tyrosine residues is a pre-requisite for ubiquitination of intactAAV2 particles, and that a substantial number of ubiquitinated virionsare recognized and degraded by cytoplasmic proteasomes on their way tothe nucleus, leading to inefficient nuclear transport.

Substitution of surface exposed tyrosine residues on AAV2 capsids thuspermits the vectors to escape ubiquitination and thus,proteasome-mediated degradation. The inventors have demonstrated thatAAV capsids can be phosphorylated at tyrosine residues by EGFR-PTK in anin vitro phosphorylation assay, and that the phosphorylated AAV capsidsretain their structural integrity. Although phosphorylated AAV vectorscould enter cells as efficiently as their unphosphorylated counterparts,their transduction efficiency was significantly reduced. This reductionwas not due to impaired viral second-strand DNA synthesis sincetransduction efficiency of both single-stranded AAV (ssAAV) andself-complementary AAV (rAAV) vectors was decreased by ˜68% and ˜74%,respectively. Intracellular trafficking of tyrosine-phosphorylated AAVvectors from cytoplasm to nucleus was also significantly decreased, mostlikely led to ubiquitination of AAV capsids followed byproteasome-mediated degradation.

In one embodiment, the invention provides a recombinant adeno-associatedviral (rAAV) vector that comprises at least a first capsid proteincomprising at least a first phosphorylated tyrosine amino acid residue,and wherein said vector further comprises at least a first nucleic acidsegment that encodes a therapeutic agent operably linked to a promotercapable of expressing said segment in a host cell that comprises saidvector.

The rAAV vector may optionally further comprise at least one enhancersequence that is operably linked to the nucleic acid segment.

Exemplary enhancer sequences include, but are not limited to, one ormore selected from the group consisting of a CMV enhancer, a syntheticenhancer, a liver-specific enhancer, an vascular-specific enhancer, abrain-specific enhancer, a neural cell-specific enhancer, alung-specific enhancer, a muscle-specific enhancer, a kidney-specificenhancer, a pancreas-specific enhancer, and an islet cell-specificenhancer.

Exemplary promoters include one or more heterologous, tissue-specific,constitutive or inducible promoters, including, for example, but notlimited to, a promoter selected from the group consisting of a CMVpromoter, a β-actin promoter, an insulin promoter, an enolase promoter,a BDNF promoter, an NGF promoter, an EGF promoter, a growth factorpromoter, an axon-specific promoter, a dendrite-specific promoter, abrain-specific promoter, a hippocampal-specific promoter, akidney-specific promoter, an elafin promoter, a cytokine promoter, aninterferon promoter, a growth factor promoter, an alpha-1 antitrypsinpromoter, a brain-specific promoter, a neural cell-specific promoter, acentral nervous system cell-specific promoter, a peripheral nervoussystem cell-specific promoter, an interleukin promoter, a serpinpromoter, a hybrid CMV promoter, a hybrid β-actin promoter, an EF1promoter, a U1a promoter, a U1b promoter, a Tet-inducible promoter and aVP16-LexA promoter. In exemplary embodiments, the promoter is amammalian or avian β-actin promoter.

The first nucleic acid segment may also further comprise apost-transcriptional regulatory sequence or a polyadenylation signal,including, for example, but not limited to, a woodchuck hepatitis viruspost-transcription regulatory element, or a polyadenylation signalsequence.

Exemplary therapeutic agents include, but are not limited to, an agentselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen binding fragment, a ribozyme, a peptide nucleicacid, an siRNA, an RNAi, an antisense oligonucleotide and an antisensepolynucleotide.

In exemplary embodiments, the rAAV vectors of the invention will encodea therapeutic protein or polypeptide selected from the group consistingof an adrenergic agonist, an anti-apoptosis factor, an apoptosisinhibitor, a cytokine receptor, a cytokine, a cytotoxin, anerythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, agrowth factor, a growth factor receptor, a hormone, a hormone receptor,an interferon, an interleukin, an interleukin receptor, a kinase, akinase inhibitor, a nerve growth factor, a netrin, a neuroactivepeptide, a neuroactive peptide receptor, a neurogenic factor, aneurogenic factor receptor, a neuropilin, a neurotrophic factor, aneurotrophin, a neurotrophin receptor, an N-methyl-D-aspartateantagonist, a plexin, a protease, a protease inhibitor, a proteindecarboxylase, a protein kinase, a protein kinsase inhibitor, aproteolytic protein, a proteolytic protein inhibitor, a semaphorin, asemaphorin receptor, a serotonin transport protein, a serotonin uptakeinhibitor, a serotonin receptor, a serpin, a serpin receptor, and atumor suppressor.

In certain applications, the modified high-transduction efficiencyvectors may comprise a nucleic acid segment that encodes a polypeptideselected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF,GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, TGF-B2,TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(I87A), viral IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18. Such therapeutic agentsmay be of human, murine, avian, porcine, bovine, ovine, feline, canine,equine, epine, caprine, lupine or primate origin.

In exemplary embodiments, the mutation may be made at one or more of thefollowing amino acid residues: Tyr252, Tyr272, Tyr444, Tyr500, Tyr700,Tyr704, Tyr730; Tyr275, Tyr281, Tyr508, Tyr576, Tyr612, Tyr673 orTyr720. Exemplary mutations are tyrosine-to-phenylalanine mutationsincluding, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F, Y704F,Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F.

The rAAV vectors of the present invention may be comprised within anadeno-associated viral particle or infectious rAAV virion, including forexample, virions selected from the group consisting of an AAV serotype1, an AAV serotype 2, an AAV serotype 3, an AAV serotype 4, an AAVserotype 5 and an AAV serotype 6.

The rAAV vectors of the present invention may also be comprised withinan isolated mammalian host cell, including for example, human, primate,murine, feline, canine, porcine, ovine, bovine, equine, epine, caprineand lupine host cells. The rAAV vectors may be comprised within anisolated mammalian host cell such as a human endothelial, epithelial,vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle,bone, neural, blood, or brain cell.

In related embodiments, the invention also provides a composition thatcomprises one or more of the disclosed tyrosine-modified rAAV vectorscomprised within a kit for diagnosing, preventing, treating orameliorating one or more symptoms of a mammalian disease, injury,disorder, trauma or dysfunction. Such kits may be useful in diagnosis,prophylaxis, and/or therapy, and particularly useful in the treatment,prevention, and/or amelioration of one or more symptoms of cancer,diabetes, autoimmune disease, kidney disease, cardiovascular disease,pancreatic disease, intestinal disease, liver disease, neurologicaldisease, neuromuscular disorder, neuromotor deficit, neuroskeletalimpairment, neurological disability, neurosensory dysfunction, stroke,ischemia, eating disorder, α₁-antitrypsin (AAT) deficiency, Batten'sdisease, Alzheimer's disease, Huntington's disease, Parkinson's disease,skeletal disease, trauma, or pulmonary disease.

The invention also provides for the use of a composition disclosedherein in the manufacture of a medicament for treating, preventing orameliorating the symptoms of a disease, disorder, dysfunction, injury ortrauma, including, but not limited to, the treatment, prevention, and/orprophylaxis of a disease, disorder or dysfunction, and/or theamelioration of one or more symptoms of such a disease, disorder ordysfunction. Exemplary conditions for which rAAV viral based genetherapy may find particular utility include, but are not limited to,cancer, diabetes, autoimmune disease, kidney disease, cardiovasculardisease, pancreatic disease, intestinal disease, liver disease,neurological disease, neuromuscular disorder, neuromotor deficit,neuroskeletal impairment, neurological disability, neurosensorydysfunction, stroke, α₁-antitrypsin (AAT) deficiency, Batten's disease,ischemia, an eating disorder, Alzheimer's disease, Huntington's disease,Parkinson's disease, skeletal disease and pulmonary disease.

The invention also provides a method for treating or ameliorating thesymptoms of such a disease, injury, disorder, or dysfunction in a mammalSuch methods generally involve at least the step of administering to amammal in need thereof, one or more of the tyrosine-modified rAAVvectors as disclosed herein, in an amount and for a time sufficient totreat or ameliorate the symptoms of such a disease, injury, disorder, ordysfunction in the mammal.

Such treatment regimens are particularly contemplated in human therapy,via administration of one or more compositions either intramuscularly,intravenously, subcutaneously, intrathecally, intraperitoneally, or bydirect injection into an organ or a tissue of the mammal under care.

The invention also provides a method for providing to a mammal in needthereof, a therapeutically-effective amount of the rAAV compositions ofthe present invention, in an amount, and for a time effective to providethe patient with a therapeutically-effective amount of the desiredtherapeutic agent(s) encoded by one or more nucleic acid segmentscomprised within the rAAV vector. Preferably, the therapeutic agent isselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen binding fragment, a ribozyme, a peptide nucleicacid, an siRNA, an RNAi, an antisense oligonucleotide and an antisensepolynucleotide.

AAV Vector Compositions

One important aspect of the present methodology is the fact that theimproved rAAV vectors described herein permit the delivery of smallertiters of viral particles in order to achieve the same transductionefficiency as that obtained using higher levels of conventional,non-surface capsid modified rAAV vectors. To that end, the amount of AAVcompositions and time of administration of such compositions will bewithin the purview of the skilled artisan having benefit of the presentteachings. In fact, the inventors contemplate that the administration oftherapeutically-effective amounts of the disclosed compositions may beachieved by a single administration, such as for example, a singleinjection of sufficient numbers of infectious particles to providetherapeutic benefit to the patient undergoing such treatment.Alternatively, in some circumstances, it may be desirable to providemultiple, or successive administrations of the AAV vector compositions,either over a relatively short, or a relatively prolonged period oftime, as may be determined by the medical practitioner overseeing theadministration of such compositions. For example, the number ofinfectious particles administered to a mammal may be on the order ofabout 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or even higher, infectiousparticles/ml given either as a single dose, or divided into two or moreadministrations as may be required to achieve therapy of the particulardisease or disorder being treated. In fact, in certain embodiments, itmay be desirable to administer two or more different AAV vectorcompositions, either alone, or in combination with one or more othertherapeutic drugs to achieve the desired effects of a particular therapyregimen. In most rAAV-based gene therapy regimens, the inventors believethat a lower titer of infectious particles will be required when usingthe modified-capsid rAAV vectors, than compared to conventional genetherapy protocols.

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous polynucleotidesegment (such as DNA segment that leads to the transcription of abiologically-active therapeutic agent) has been introduced. Therefore,engineered cells are distinguishable from naturally occurring cells,which do not contain a recombinantly introduced exogenous DNA segment.Engineered cells are, therefore, cells that comprise at least one ormore heterologous polynucleotide segments introduced through the hand ofman.

To express a therapeutic agent in accordance with the present inventionone may prepare a tyrosine-modified rAAV expression vector thatcomprises a therapeutic agent-encoding nucleic acid segment under thecontrol of one or more promoters. To bring a sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame generally between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.The “upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise an rAAV vector.Such vectors are described in detail herein.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, and/orantisense oligonucleotides, to a particular cell transfected with thevector, one may employ the rAAV vectors or the tyrosine-modified rAAVvectors disclosed herein by genetically modifying the vectors to furthercomprise at least a first exogenous polynucleotide operably positioneddownstream and under the control of at least a first heterologouspromoter that expresses the polynucleotide in a cell comprising thevector to produce the encoded peptide, protein, polypeptide, ribozyme,siRNA, RNAi or antisense oligonucleotide. Such constructs may employheterologous promoters that are constitutive, inducible, or evencell-specific promoters. Exemplary such promoters include, but are notlimited to, viral, mammalian, and avian promoters, including for examplea CMV promoter, a β-actin promoter, a hybrid CMV promoter, a hybridβ-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter, aTet-inducible promoter, a VP16-LexA promoter, and such like.

The vectors or expression systems may also further comprise one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise at least a first CMV enhancer, a synthetic enhancer, ora cell- or tissue-specific enhancer. The exogenous polynucleotide mayalso further comprise one or more intron sequences.

Pharmaceutical Compositions

The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.The rAAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders, and in particular,articular diseases, disorders, and dysfunctions, including for exampleosteoarthritis, rheumatoid arthritis, and related disorders. Moreover,pharmaceutical compositions comprising one or more of the nucleic acidcompounds disclosed herein, provide significant advantages over existingconventional therapies—namely, (1) their reduced side effects, (2) theirincreased efficacy for prolonged periods of time, (3) their ability toincrease patient compliance due to their ability to provide therapeuticeffects following as little as a single administration of the selectedtherapeutic rAAV composition to affected individuals. Exemplarypharmaceutical compositions and methods for their administration arediscussed in significant detail hereinbelow.

The invention also provides compositions comprising one or more of thedisclosed rAAV vectors, expression systems, virions, viral particles; ormammalian cells. As described hereinbelow, such compositions may furthercomprise a pharmaceutical excipient, buffer, or diluent, and may beformulated for administration to an animal, and particularly a humanbeing. Such compositions may further optionally comprise a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere, ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a mammal in need thereof. Suchcompositions may be formulated for use in a variety of therapies, suchas for example, in the amelioration, prevention, and/or treatment ofconditions such as peptide deficiency, polypeptide deficiency, peptideoverexpression, polypeptide overexpression, including for example,conditions which result in diseases or disorders such as cancers,tumors, or other malignant growths, neurological deficit dysfunction,autoimmune diseases, articular diseases, cardiac or pulmonary diseases,ischemia, stroke, cerebrovascular accidents, transient ischemic attacks(TIA); diabetes and/or other diseases of the pancreas; cardiocirculatorydisease or dysfunction (including, e.g., hypotension, hypertension,atherosclerosis, hypercholesterolemia, vascular damage or disease;neural diseases (including, e.g., Alzheimer's, Huntington's, Tay-Sach'sand Parkinson's disease, memory loss, trauma, motor impairment,neuropathy, and related disorders); biliary, renal or hepatic disease ordysfunction; musculoskeletal or neuromuscular diseases (including, e.g.,arthritis, palsy, cystic fibrosis (CF), amyotrophic lateral sclerosis(ALS), multiple sclerosis (MS), muscular dystrophy (MD), and such like).

In certain embodiments, the present invention concerns formulation ofone or more rAAV-based compositions disclosed herein in pharmaceuticallyacceptable solutions for administration to a cell or an animal, eitheralone or in combination with one or more other modalities of therapy,and in particular, for therapy of human cells, tissues, and diseasesaffecting man.

It will also be understood that, if desired, nucleic acid segments, RNA,DNA or PNA compositions that express one or more of therapeutic geneproducts may be administered in combination with other agents as well,such as, e.g., proteins or polypeptides or variouspharmaceutically-active agents, including one or more systemic ortopical administrations of therapeutic polypeptides, biologically activefragments, or variants thereof. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The rAAV-based genetic compositions maythus be delivered along with various other agents as required in theparticular instance. Such compositions may be purified from host cellsor other biological sources, or alternatively may be chemicallysynthesized as described herein. Likewise, such compositions may furthercomprise substituted or derivatized RNA, DNA, siRNA, mRNA, tRNA,ribozyme, catalytic RNA molecules, or PNA compositions and such like.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal,intra-articular, intramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraocularly, intravitreally, parenterally, subcutaneously,intravenously, intracerebro-ventricularly, intramuscularly,intrathecally, orally, intraperitoneally, by oral or nasal inhalation,or by direct injection to one or more cells, tissues, or organs bydirect injection. The methods of administration may also include thosemodalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as freebase orpharmacologically acceptable salts may be prepared in sterile water andmay also suitably mixed with one or more surfactants, such ashydroxypropylcellulose. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms of the AAV-based viral compositions suitablefor injectable use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions (U.S. Pat. No. 5,466,468, specificallyincorporated herein by reference in its entirety). In all cases the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(e.g., glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and/or vegetable oils. Properfluidity may be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialad antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activeAAV vector-delivered therapeutic polypeptide-encoding DNA fragments inthe required amount in the appropriate solvent with several of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

The amount of AAV compositions and time of administration of suchcompositions will be within the purview of the skilled artisan havingbenefit of the present teachings. It is likely, however, that theadministration of therapeutically-effective amounts of the disclosedcompositions may be achieved by a single administration, such as forexample, a single injection of sufficient numbers of infectiousparticles to provide therapeutic benefit to the patient undergoing suchtreatment. Alternatively, in some circumstances, it may be desirable toprovide multiple, or successive administrations of the AAV vectorcompositions, either over a relatively short, or a relatively prolongedperiod of time, as may be determined by the medical practitioneroverseeing the administration of such compositions. For example, thenumber of infectious particles administered to a mammal may be on theorder of about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or even higher,infectious particles/ml given either as a single dose, or divided intotwo or more administrations as may be required to achieve therapy of theparticular disease or disorder being treated. In fact, in certainembodiments, it may be desirable to administer two or more different AAVvector compositions, either alone, or in combination with one or moreother therapeutic drugs to achieve the desired effects of a particulartherapy regimen.

Expression Vectors

The present invention contemplates a variety of AAV-based expressionsystems, and vectors. In one embodiment the preferred AAV expressionvectors comprise at least a first nucleic acid segment that encodes atherapeutic peptide, protein, or polypeptide. In another embodiment, thepreferred AAV expression vectors disclosed herein comprise at least afirst nucleic acid segment that encodes an antisense molecule. Inanother embodiment, a promoter is operatively linked to a sequenceregion that encodes a functional mRNA, a tRNA, a ribozyme or anantisense RNA.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depend directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the functional RNA to which itis operatively linked.

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

Therapeutic and Diagnostic Kits

The invention also encompasses one or more of the genetically-modifiedrAAV vector compositions described herein together with one or morepharmaceutically-acceptable excipients, carriers, diluents, adjuvants,and/or other components, as may be employed in the formulation ofparticular rAAV-polynucleotide delivery formulations, and in thepreparation of therapeutic agents for administration to a mammal, and inparticularly, to a human. In particular, such kits may comprise one ormore of the disclosed rAAV compositions in combination with instructionsfor using the viral vector in the treatment of such disorders in amammal, and may typically further include containers prepared forconvenient commercial packaging.

As such, preferred animals for administration of the pharmaceuticalcompositions disclosed herein include mammals, and particularly humans.Other preferred animals include murines, bovines, equines, porcines,canines, and felines. The composition may include partially orsignificantly purified rAAV compositions, either alone, or incombination with one or more additional active ingredients, which may beobtained from natural or recombinant sources, or which may be obtainablenaturally or either chemically synthesized, or alternatively produced invitro from recombinant host cells expressing DNA segments encoding suchadditional active ingredients.

Therapeutic kits may also be prepared that comprise at least one of thecompositions disclosed herein and instructions for using the compositionas a therapeutic agent. The container means for such kits may typicallycomprise at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which the disclosed rAAV composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticpolypeptide composition is also provided, the kit may also contain asecond distinct container means into which this second composition maybe placed. Alternatively, the plurality of therapeutic biologicallyactive compositions may be prepared in a single pharmaceuticalcomposition, and may be packaged in a single container means, such as avial, flask, syringe, bottle, or other suitable single container means.The kits of the present invention will also typically include a meansfor containing the vial(s) in close confinement for commercial sale,such as, e.g., injection or blow-molded plastic containers into whichthe desired vial(s) are retained.

rAAV Capsid Proteins

Supramolecular assembly of 60 individual capsid protein subunits into anon-enveloped, T-1 icosahedral lattice capable of protecting a 4.7-kbsingle-stranded DNA genome is a critical step in the life-cycle of thehelper-dependent human parvovirus, adeno-associated virus2 (AAV2). Themature 20-nm diameter AAV2 particle is composed of three structuralproteins designated VP1, VP2, and VP3 (molecular masses of 87, 73, and62 kDa respectively) in a ratio of 1:1:18. Based on its symmetry andthese molecular weight estimates, of the 60 capsid proteins comprisingthe particle, three are VP1 proteins, three are VP2 proteins, andfifty-four are VP3 proteins. The employment of three structural proteinsmakes AAV serotypes unique among parvoviruses, as all others knownpackage their genomes within icosahedral particles composed of only twocapsid proteins. The anti-parallel β-strand barreloid arrangement ofthese 60 capsid proteins results in a particle with a defined tropismthat is highly resistant to degradation. Modification of one or moretyrosine residues in one or more of the capsid proteins has been shownby the inventors to achieve superior transfection at lower dose andlower cost than conventional protocols. By site-specifically modifyingone or more tyrosine residues on the surface of the capsid, theinventors have achieved significant improvement in transductionefficiency.

Nucleic Acid Amplification

In certain embodiments, it may be necessary to employ one or morenucleic acid amplification techniques to produce the nucleic acidsegments of the present invention. Various methods are well-known toartisans in the field, including for example, those techniques describedherein: Nucleic acid, used as a template for amplification, may beisolated from cells contained in the biological sample according tostandard methodologies (Sambrook et al., 1989). The nucleic acid may begenomic DNA or fractionated or whole cell RNA. Where RNA is used, it maybe desired to convert the RNA to a complementary DNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to the ribozymes or conserved flanking regions arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred. Once hybridized, the nucleic acid:primer complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (e.g., Affymax® technology). A number of templatedependent processes are available to amplify the marker sequencespresent in a given template sample. One of the best-known amplificationmethods is the polymerase chain reaction (referred to as PCR™), which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159(each of which is incorporated herein by reference in its entirety).

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal. (1989). Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin Int. Pat. Appl. Publ. No. WO 90/07641 (specifically incorporatedherein by reference). Polymerase chain reaction methodologies are wellknown in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, and incorporated herein by reference inits entirety. In LCR, two complementary probe pairs are prepared, and inthe presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qβ Replicase (QβR), described in Int. Pat. Appl. No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA), described in U.S. Pat. Nos.5,455,166, 5,648,211, 5,712,124 and 5,744,311, each incorporated hereinby reference, is another method of carrying out isothermal amplificationof nucleic acids which involves multiple rounds of strand displacementand synthesis, i.e., nick translation. A similar method, called RepairChain Reaction (RCR), involves annealing several probes throughout aregion targeted for amplification, followed by a repair reaction inwhich only two of the four bases are present. The other two bases can beadded as biotinylated derivatives for easy detection. A similar approachis used in SDA. Target specific sequences can also be detected using acyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′ sequencesof non-specific DNA and a middle sequence of specific RNA is hybridizedto DNA that is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products that are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

Still another amplification methods described in GB Application No. 2202 328, and in Int. Pat. Appl. No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., Int. Pat. Appl. Publ. No.WO 88/10315, incorporated herein by reference. In NASBA, the nucleicacids can be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer that has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., Int. Pat. Appl. Publ. No. WO 89/06700 (incorporatedherein by reference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990, specifically incorporated herein by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (see e.g., Sambrook et al., 1989).

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

Biological Functional Equivalents

Modification and changes to the structure of the polynucleotides andpolypeptides of wild-type rAAV vectors to provide the improved rAAVvirions as described in the present invention to obtain functional viralvectors that possess desirable characteristics, particularly withrespect to improved delivery of therapeutic gene constructs to selectedmammalian cell, tissues, and organs for the treatment, prevention, andprophylaxis of various diseases and disorders, as well as means for theamelioration of symptoms of such diseases, and to facilitate theexpression of exogenous therapeutic and/or prophylactic polypeptides ofinterest via rAAV vector-mediated gene therapy. As mentioned above, oneof the key aspects of the present invention is the creation of one ormore mutations into specific polynucleotide sequences that encode one ormore of the therapeutic agents encoded by the disclosed rAAV constructs.In certain circumstances, the resulting polypeptide sequence is alteredby these mutations, or in other cases, the sequence of the polypeptideis unchanged by one or more mutations in the encoding polynucleotide toproduce modified vectors with improved properties for effecting genetherapy in mammalian systems.

When it is desirable to alter the amino acid sequence of a polypeptideto create an equivalent, or even an improved, second-generationmolecule, the amino acid changes may be achieved by changing one or moreof the codons of the encoding DNA sequence, according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the polynucleotide sequences disclosed herein,without appreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take several of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Exemplary Definitions

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from natural sources, chemicallysynthesized, modified, or otherwise prepared or synthesized in whole orin part by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

A, an: In accordance with long standing patent law convention, the words“a” and “an” when used in this application, including the claims,denotes “one or more.”

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a polynucleotide such as astructural gene to synthesize the encoded peptide or polypeptide.

Promoter: a term used to generally describe the region or regions of anucleic acid sequence that regulates transcription.

Regulatory Element: a term used to generally describe the region orregions of a nucleic acid sequence that regulates transcription.Exemplary regulatory elements include, but are not limited to,enhancers, post-transcriptional elements, transcriptional controlsequences, and such like.

Structural gene: A polynucleotide, such as a gene, that is expressed toproduce an encoded peptide, polypeptide, protein, ribozyme, catalyticRNA molecule, siRNA, or antisense molecule.

Transformation: A process of introducing an exogenous polynucleotidesequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNAmolecule) into a host cell or protoplast in which the exogenouspolynucleotide is incorporated into at least a first chromosome or iscapable of autonomous replication within the transformed host cell.Transfection, electroporation, and “naked” nucleic acid uptake allrepresent examples of techniques used to transform a host cell with oneor more polynucleotides.

Transformed cell: A host cell whose nucleic acid complement has beenaltered by the introduction of one or more exogenous polynucleotidesinto that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell, or from the progeny or offspring ofany generation of such a transformed host cell.

Vector: A nucleic acid molecule (typically comprised of DNA) capable ofreplication in a host cell and/or to which another nucleic acid segmentcan be operatively linked so as to bring about replication of theattached segment. A plasmid, cosmid, or a virus is an exemplary vector.

The terms “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

Desirably, which highly homologous fragments are desired, the extent ofpercent identity between the two sequences will be at least about 80%,preferably at least about 85%, and more preferably about 90% or 95% orhigher, as readily determined by one or more of the sequence comparisonalgorithms well-known to those of skill in the art, such as e.g., theFASTA program analysis described by Pearson and Lipman (Proc. Natl.Acad. Sci. USA, 85(8):2444-8, April 1988).

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by the hand of man in alaboratory is naturally-occurring. As used herein, laboratory strains ofrodents that may have been selectively bred according to classicalgenetics are considered naturally occurring animals.

As used herein, a “heterologous” is defined in relation to apredetermined referenced gene sequence. For example, with respect to astructural gene sequence, a heterologous promoter is defined as apromoter which does not naturally occur adjacent to the referencedstructural gene, but which is positioned by laboratory manipulation.Likewise, a heterologous gene or nucleic acid segment is defined as agene or segment that does not naturally occur adjacent to the referencedpromoter and/or enhancer elements.

“Transcriptional regulatory element” refers to a polynucleotide sequencethat activates transcription alone or in combination with one or moreother nucleic acid sequences. A transcriptional regulatory element can,for example, comprise one or more promoters, one or more responseelements, one or more negative regulatory elements, and/or one or moreenhancers.

As used herein, a “transcription factor recognition site” and a“transcription factor binding site” refer to a polynucleotidesequence(s) or sequence motif(s) which are identified as being sites forthe sequence-specific interaction of one or more transcription factors,frequently taking the form of direct protein-DNA binding. Typically,transcription factor binding sites can be identified by DNAfootprinting, gel mobility shift assays, and the like, and/or can bepredicted on the basis of known consensus sequence motifs, or by othermethods known to those of skill in the art.

As used herein, the term “operably linked” refers to a linkage of two ormore polynucleotides or two or more nucleic acid sequences in afunctional relationship. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the coding sequence.“Operably linked” means that the nucleic acid sequences being linked aretypically contiguous, or substantially contiguous, and, where necessaryto join two protein coding regions, contiguous and in reading frame.However, since enhancers generally function when separated from thepromoter by several kilobases and intronic sequences may be of variablelengths, some polynucleotide elements may be operably linked but notcontiguous.

“Transcriptional unit” refers to a polynucleotide sequence thatcomprises at least a first structural gene operably linked to at least afirst cis-acting promoter sequence and optionally linked operably to oneor more other cis-acting nucleic acid sequences necessary for efficienttranscription of the structural gene sequences, and at least a firstdistal regulatory element as may be required for the appropriatetissue-specific and developmental transcription of the structural genesequence operably positioned under the control of the promoter and/orenhancer elements, as well as any additional cis sequences that arenecessary for efficient transcription and translation (e.g.,polyadenylation site(s), mRNA stability controlling sequence(s), etc.

The term “substantially complementary,” when used to define either aminoacid or nucleic acid sequences, means that a particular subjectsequence, for example, an oligonucleotide sequence, is substantiallycomplementary to all or a portion of the selected sequence, and thuswill specifically bind to a portion of an mRNA encoding the selectedsequence. As such, typically the sequences will be highly complementaryto the mRNA “target” sequence, and will have no more than about 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementaryportion of the sequence. In many instances, it may be desirable for thesequences to be exact matches, i.e. be completely complementary to thesequence to which the oligonucleotide specifically binds, and thereforehave zero mismatches along the complementary stretch. As such, highlycomplementary sequences will typically bind quite specifically to thetarget sequence region of the mRNA and will therefore be highlyefficient in reducing, and/or even inhibiting the translation of thetarget mRNA sequence into polypeptide product.

Substantially complementary oligonucleotide sequences will be greaterthan about 80 percent complementary (or ‘% exact-match’) to thecorresponding mRNA target sequence to which the oligonucleotidespecifically binds, and will, more preferably be greater than about 85percent complementary to the corresponding mRNA target sequence to whichthe oligonucleotide specifically binds. In certain aspects, as describedabove, it will be desirable to have even more substantiallycomplementary oligonucleotide sequences for use in the practice of theinvention, and in such instances, the oligonucleotide sequences will begreater than about 90 percent complementary to the corresponding mRNAtarget sequence to which the oligonucleotide specifically binds, and mayin certain embodiments be greater than about 95 percent complementary tothe corresponding mRNA target sequence to which the oligonucleotidespecifically binds, and even up to and including 96%, 97%, 98%, 99%, andeven 100% exact match complementary to all or a portion of the targetmRNA to which the designed oligonucleotide specifically binds.

Percent similarity or percent complementary of any of the disclosedsequences may be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0, available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (J. Mol.Biol., 48(3):443-53, 1970). Briefly, the GAP program defines similarityas the number of aligned symbols (i.e., nucleotides or amino acids) thatare similar, divided by the total number of symbols in the shorter ofthe two sequences. The preferred default parameters for the GAP programinclude: (1) a unary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) for nucleotides, and the weightedcomparison matrix of Gribskov and Burgess (Nucl. Acids Res.,14:6745-6763, 1986), (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human, and in particular, whenadministered to the human eye. The preparation of an aqueous compositionthat contains a protein as an active ingredient is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified.

As used herein, the term “operatively linked” means that a promoter isconnected to a functional RNA in such a way that the transcription ofthat functional RNA is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a functional RNA are well known inthe art.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 AAV2-Mediated Gene Transfer: A Dual Role of EGFR ProteinTyrosine Kinase Signaling in Ubiquitination of Viral Capsids and ViralSecond-Strand DNA Synthesis

The adeno-associated virus 2 (AAV2), a non-pathogenic human parvovirus,has gained attention as an alternative to the more commonly usedretrovirus- and adenovirus-based vectors for gene transfer and genetherapy^(1, 2). Recombinant AAV2 vectors are currently in use in PhaseI/II clinical trials for gene therapy of a number of diseases such ascystic fibrosis, α-1 antitrypsin deficiency, Parkinson's disease,Batten's disease, and muscular dystrophy,^(3, 4, 5) and have been shownto transduce a wide variety of cells and tissues in vitro and invivo.^(2, 6-8) Systematic studies have been exploited to elucidate someof the fundamental steps in the life cycle of AAV vectors, which includeviral binding, entry,⁹⁻¹³ intracellular trafficking,¹⁴⁻¹⁷uncoating,^(18, 19) second-strand DNA synthesis,²⁰⁻²⁸ and viral genomeintegration into host cell chromosome.^(29, 30)

Two independent laboratories have described that the viral second-strandDNA synthesis is a rate-limiting step, which accounts for inefficienttransduction of certain cell types by AAV vectors.^(20, 21) Theinventors have also demonstrated that a cellular protein (designatedFKBP52), which interacts with the single-stranded D-sequence in the AAV2inverted terminal repeat (ITR), is phosphorylated at tyrosine residuesby the epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK), and inhibits the viral second-strand DNA synthesis leadingto inefficient transgene expression,²⁴ It has also been documented thatFKBP52 is dephosphorylated at tyrosine residues by T-cell proteintyrosine phosphatase (TC-PTP), which negatively regulates EGFR-PTKsignaling, leading to efficient viral second-strand DNA synthesis.²⁵Tyrosine-dephosphorylation of FKBP52 in TC-PTP-transgenic (TC-PTP TG)mice, and removal of FKBP52 in FKBP52 knockout (FKBP52-KO) mice alsolead to efficient AAV2 transduction of murine hepatocytes in vivo.²⁷

An additional rate-limiting step in AAV-mediated transduction, viralintracellular trafficking, has also become apparent, and is beingstudied extensively. The ubiquitin-proteasome pathway has been shown toplay an essential role in this step. AAV2 is likely to be degraded if itfails to escape the late endosome. If the virus escapes into thecytoplasm perinuclearly, it may be ubiquitinated and degraded by thecytoplasmic proteasome.^(16, 31-32) In previous studies with murinefibroblast,^(14, 15) it was documented that AAV2 vectors failed totraffic to the nucleus efficiently, but over-expression of TC-PTP inTC-PTP-TG mice facilitated this process.¹⁹ These studies suggested thatTC-PTP and/or EGFR-PTK signaling were involved in AAV2 intracellulartrafficking.

In the present studies, the role of EGFR-PTK signaling inubiquitination, intracellular trafficking, and AAV-mediated transgeneexpression was systematically examined. It was demonstrated that inaddition to augmenting viral second-strand DNA synthesis, perturbationsin EGFR-PTK signaling affects AAV2 transduction efficiency byfacilitating intracellular trafficking from cytoplasm to nucleus. Sincethe free ubiquitin content within a cell that regulates lysosomaldegradation of EGFR, with proteasome inhibitors affect receptorendocytosis,³³ proteasome inhibitors augment AAVtransduction,^(16, 31-35) and protein phosphorylation modulatesubiquitination of cellular and viral proteins,³⁶⁻⁴² evidence ispresented documenting that inhibition of EGFR-PTK signaling decreasesubiquitination of AAV2 capsid proteins, suggesting that ubiquitinationfollowed by proteasome-mediated degradation of AAV2 capsid proteins isalso affected by EGFR-PTK. These studies suggest that complexinteractions between EGFR-PTK signaling and ubiquitin/proteasome pathwayplay a role in AAV-mediated transduction, which is likely to beimportant in yielding new insights in the optimal use of recombinant AAVvectors in human gene therapy.

Materials and Methods

Cells, viruses, plasmids, antibodies, and chemicals. The human cervicalcarcinoma cell line, HeLa, was obtained from the American Type CultureCollection (ATCC, Rockville, Md., USA), and maintained as monolayercultures in Iscove's-modified Dulbecco's medium (IMDM) supplemented with10% newborn calf serum (NCS) and 1% (by volume) of 100× stock solutionof antibiotics (10,000 U penicillin+10,000 □μg streptomycin).Highly-purified stocks of ss recombinant AAV2 vectors containing theβ-galactosidase (lacZ) reporter gene, or red fluorescence protein (RFP)gene, or ss and sc recombinant AAV2 vectors containing enhanced greenfluorescence protein (EGFP) gene driven by the cytomegalovirus (CMV)immediate-early promoter (ssAAV2-lacZ, ssAAV2-RFP, ssAAV2-EGFP orscAAV2-EGFP) were generated as described previously.⁴⁹

Physical particle titers of recombinant vector stocks were determined byquantitative DNA slot blot analysis. Horseradish peroxidase(HRP)-conjugated antibody specific for ubiquitin (Ub) (mouse monoclonalimmunoglobulin G₁ [IgG₁], clone P4D1), and normal mouse IgG₁ werepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).Antibodies specific for intact AAV2 particles (mouse monoclonal IgG₁,clone A20) was obtained from Research Diagnostics, Inc., (Flanders,N.J., USA). MG132 was purchased from Calbiochem (La Jolla, Calif., USA),and all other chemicals used were purchased from Sigma-Aldrich Co. (St.Louis, Mo. USA).

Recombinant AAV vector transduction assay. Approximately 1×10⁵ HeLacells were plated in each well in 12-well plates and incubated at 37° C.for 12 hr. Cells were washed once with IMDM and then infected at 37° C.for 2 hr with 5×10³ particles per cell of recombinant AAV2-lacZ,ssAAV2-EGFP or scAAV2-EGFP vectors as described previously.^(24, 26, 28)Cells were incubated in complete IMDM containing 10% NCS and 1%antibiotics for 48 hr. For lacZ expression, cells were fixed and stainedwith X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Thetransduction efficiency was measured by GFP imaging using a LEICA DMIRB/E fluorescence microscope (Leica Microsystems Wetzlar GmbH,Germany). Images from three visual fields of mock-infected andvector-infected HeLa cells at 48 hr post-injection were analyzedquantitatively by ImageJ analysis software (NIH, Bethesda, Md., USA).Transgene expression was assessed as total area of green fluorescence(pixel²) per visual field (mean±SD). Analysis of variance (ANOVA) wasused to compare between test results and the control and they weredetermined to be statistically significant.

Isolation of nuclear and cytoplasmic fractions from HeLa cells. Nuclearand cytoplasmic fractions from HeLa cells were isolated as describedpreviously.¹⁹ Cells were mock-infected or infected with recombinantAAV2-lacZ vectors were washed twice with PBS 12 hr post-infection. Cellswere treated with 0.01% trypsin and washed extensively with PBS toremove any adsorbed and unadsorbed virus particles. Cell pellets weregently resuspended in 200 μl hypotonic buffer (10 mM HEPES, pH 7.9. 1.5mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF) and incubated on ice for 5min, after which 10 μl 10% NP-40 was added to each tube for ˜3 min, andobserved under a light microscope. Samples were mixed gently andcentrifuged for 5 min at 500 rpm at 4° C. Supernatants (cytoplasmicfractions) were decanted and stored on ice. Pellets (nuclear fractions)were washed twice with 1 ml hypotonic buffer and stored on ice. Thepurity of each fraction was determined to be >95%, as measured by theabsence of acid phosphatase activity (nuclear fractions) and absence ofhistone H3 (cytoplasmic fractions) as described previously.^(14, 19)

Southern blot analysis for AAV trafficking. Low M_(r) DNA samples fromnuclear and cytoplasmic fractions were isolated and electrophoresed on1% agarose gels or 1% alkaline-agarose gels followed by Southern blothybridization using a ³²P-labeled lacZ-specific DNA probe as describedpreviously.^(14, 19) Densitometric scanning of autoradiographs for thequantitation was evaluated with ImageJ® analysis software (NationalInstitutes of Health, Bethesda, Md., USA).

Preparation of whole cell lysates (WCL) and co-immunoprecipitation. WCLwere prepared as described previously,^(17, 26, 50) with the followingmodifications: briefly, 2×10⁶ HeLa cells were either mock-treated, ortreated with 500 mM Tyr23, 4 mM MG132, or both (treatment with MG132 for2 hr and then with Try23 for an additional 2 hr) for 4 hr. Cells weremock-infected or infected with ssAAV-RFP vectors at 10⁴ particles/cellfor 2 hr at 37° C. Mock-transfected cells and cells stably transfectedwith wt- or mTC-PTP expression plasmids were treated with MG132 and alsosubjected to mock-infection or infection with ssAAV-RFP vectors.

For cellular protein analyses, treated or mock-treated cells were lysedon ice in cell lysis buffer (1% Triton X-100®, 10% glycerol, 50 mMHEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA) containing 1 mMDTT, 10 mM NaF, 2 mM Na₃VO₄, 0.5 mM PMSF, 10 mg/ml aprotinin, 10 mg/mlleupeptin and 10 mg/ml pepstatin. For immunoprecipitation, cells weretreated with 0.01% trypsin and washed extensively with PBS to remove anyadsorbed and unadsorbed virus particles after treatment or at 4 hrpost-infection and then resuspended in 2 ml hypotonic buffer (20 mMHEPES pH 7.5, 5 mM KCl, 0.5 mM MgCl₂) containing 1 mM DTT, 10 mM NaF, 2mM Na₃VO₄, 0.5 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 10mg/ml. WCL was prepared by homogenization in a tight-fitting Dualltissue grinder until about 95% cell lysis was achieved as monitored bytrypan blue dye exclusion assay. WCL were cleared of non-specificbinding by incubation with 0.25 mg of normal mouse IgG₁ together with 20ml of protein G-agarose beads for 60 min at 4° C. in an orbital shaker.

After preclearing, 2 mg of capsid antibody against intact AAV2 particles(A20) (mouse IgG₁) or 2 mg of normal mouse IgG₁ (as a negative control)was added and incubated at 4° C. for 1 hr, followed by precipitationwith protein G-agarose beads at 4° C. for 12 hr in a shaker. Pelletswere collected by centrifugation at 2,500 rpm for 5 min at 4° C. andwashed four times with PBS. After the final wash, supernatants wereaspirated and discarded, and pellets were resuspended in equal volume of2×SDS sample buffer. Twenty ml of resuspended pellet solutions were usedfor Western blotting with HRP-conjugated anti-Ub antibody as describedbelow.

Western blot analyses. Western blotting was performed as describedpreviously.^(17, 26, 50) For cellular protein analyses, equivalentamounts (˜5 mg) WCL samples were separated by 10% SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to Immobilon-P® membranes(Millipore, Bedford, Mass., USA). For immunoprecipitation, resuspendedpellet solutions were boiled for 2-3 min and 20 ml of samples were usedfor SDS-PAGE. Membranes were blocked at 4° C. for 12 hr with 5% nonfatmilk in 1× Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mMNaCl). Membranes were treated with monoclonal HRP-conjugated anti-Ubantibody (1:2,000 dilution). Immunoreactive bands were visualized usingchemiluminescence (ECL-Plus™, Amersham Pharmacia Biotech, Piscataway,N.J., USA).

Results

Inhibition of EGFR-PTK signaling increases EGFP transgene expressionfollowing transduction with both ssAAV2 and scAAV2 vectors. Inpreviously-published studies,^(23-25, 27, 43) the inventors and theircollaborators have documented that the inhibition of EGFR-PTK signalingleads to dephosphorylation of FKBP52 at tyrosine residues, andfacilitates viral second-strand DNA synthesis resulting in efficienttransgene expression. Since double-stranded scAAV2 vectors,^(44, 45)which bypass the requirement for second-strand DNA synthesis, achievemuch higher transduction efficiency, the following predication wasassessed: scAAV2-mediated transgene expression should not be influencedby the inhibition of EGFR-PTK signaling if viral second-strand DNAsynthesis is the sole mechanism involved. In the first set of studies,HeLa cells were treated with Tyr23, a specific inhibitor of EGFR-PTK⁴³,transduced with recombinant ssAAV2-EGFP or scAAV2-EGFP vectors, andtransgene expression was determined 48 hr post-transduction.

From the results shown in FIG. 1A, it is evident that whereasmock-infected HeLa cells showed no green fluorescence, only ˜3% of HeLacells transduced with the ssAAV2-EGFP vector were EGFP-positive, andTyr23 treatment led to ˜12-fold increase in ssAAV transductionefficiency (FIG. 1B), consistent with earlier results^(25, 27, 43).Although the transduction efficiency of rAAV vectors was ˜4-fold highercompared with that of their single-stranded counterparts, as expected,but surprisingly, Tyr23 treatment also led to a further ˜10-foldincrease in the transduction efficiency of rAAV vectors (FIG. 1B).

This increase was not due to contamination of rAAV vectors with ssAAVvectors, the generation of which has been recently documented⁴⁶. Thesedata, nonetheless, suggested that perturbations in EGFR signaling affectadditional aspects of AAV-mediated transduction beyond viralsecond-strand DNA synthesis.

Since stable transfection with a TC-PTP expression plasmid leads toinhibition of EGFR-PTK signaling and efficient transgene expressionmediated by ssAAV vectors²⁵, it was reasoned that deliberateover-expression of TC-PTP would also lead to a significant increase intransduction efficiency of scAAV2 vectors. HeLa cells were eithermock-transfected or stably transfected with the wild-type (wt)- or a C-Smutant (m)-TC-PTP expression plasmid, and were infected with ssAAV2-EGFPor scAAV2-EGFP vectors, and transgene expression was visualized 48-hrs'post-infection. As can be seen in FIG. 2A, whereas mock-infected HeLacells showed no green fluorescence, and only ˜3% of mock-transfectedcells transduced with ssAAV-EGFP vector were EGFP-positive, asignificantly increase (˜15-fold) in transduction efficiency of ssAAV2vectors in cells stably transfected with the wtTC-PTP expression plasmidwas obtained, consistent with previously published reports^(22, 27).This increase was not observed when the mTC-PTP expression plasmid wasused.

It is noteworthy that although the transduction efficiency of scAAV2vectors in HeLa cells is ˜8-fold higher compared with their sscounterparts, stably transfection with the wtTC-PTP expression plasmidleads to a further ˜10-fold increase (FIG. 2B). These data corroboratethat inhibition of EGFR-PTK signaling by pre-treatment with Tyr23 orover-expression of TC-PTP augments AAV2 transduction involves othermechanism(s) in addition to facilitating viral second-strand DNAsynthesis.

Nuclear transport of AAV is improved following pre-treatment with Tyr23,over-expression of wtTC-PTP, or proteasome inhibitor, MG132. It waspreviously documented that over-expression of TC-PTP in TC-PTP-TG micefacilitated AAV2 vector transport to the nucleus in primary murinehematopoietic cells,¹⁹ which suggested that EGFR-PTK signaling mightalso be involved in AAV trafficking. To further examine this hypothesis,the fate of the input viral DNA was examined in cells treated withTyr23, or stably transfected with the wtTC-PTP. Mock-treated cells,cells stably transfected with mTC-PTP, and cells treated with MG132, aspecific inhibitor of proteasome,^(16, 31, 32) known to augment AAVnuclear transport,^(16, 34, 35) were used as appropriate controls.Nuclear and cytoplasmic fractions were obtained 12 hr post-infection,low M_(r) DNA was isolated from these fractions, and wereelectrophoresed on 1% agarose gels followed by Southern blot analysis(FIG. 3A) and densitometric scanning of autoradiographs (FIG. 3B).

As is evident, ˜64% of the input ssAAV DNA was present in thecytoplasmic fraction in control cells (lane 1). Consistent withpreviously published studies^(16, 34, 35), pre-treatment with MG132improved AAV2 trafficking to the nucleus up to ˜62% (lane 10).Interestingly, in cells pre-treated with Tyr23, or stably transfectedwith the wtTC-PTP, the input ssAAV2 DNA in the nuclear fraction wasincreased to ˜52% and ˜54%, respectively (lanes 4 and 8). In cellstransfected with the mTC-PTP, on the other hand, only ˜38% of the inputssAAV DNA was present in the nuclear fraction (lane 6), which wassimilar to that in control cells (lane 2). The possibility that Tyr23and TC-PTP affect transcriptional and translational events to increasetransgene expression, as they do not improve nuclear delivery of AAV aswell as MG132, was ruled out by plasmid DNA-mediated transfection ofHeLa cells in which neither treatment with Tyr23, nor over-expression ofTC-PTP, showed any increase in transgene expression (FIG. 9). Theseresults further document that inhibition of EGFR-PTK signalingfacilitates nuclear transport of AAV vectors.

Transduction efficiency of both ssAAV and rAAV vectors in cellsover-expressing TC-PTP, or following pre-treatment with Tyr23, is notfurther enhanced by MG132. It was then examined whether inhibition ofEGFR-PTK signaling by treatment with Tyr23, or over-expression ofTC-PTP, modulates the ubiquitin/proteasome pathway involved in AAV2transduction, because the free ubiquitin content within a cell thatregulates lysosomal degradation of EGFR, and proteasome inhibitors havebeen implicated in the regulation of EGFR Endocytosis,³³ proteasomeinhibitors have been shown to augment AAVtransduction,^(16, 31, 32, 34, 35) and protein phosphorylation has beenimplicated in the regulation of ubiquitination of cellular and viralproteins.³⁶⁻⁴² Cells were mock-treated or treated with Tyr23, MG132 orboth, or either stably transfected with wt- or mTC-PTP expressionplasmids were either mock-treated or treated with MG132. All treatedcells and appropriate controls were infected with recombinantssAAV2-lacZ or scAAV2-EGFP vectors, and transgene expression wasdetermined 48 hrs post-transduction. These results are shown in FIG. 4A.

Consistent with previously published studies,^(23-25, 27, 43) >5% ofcells transduced with ssAAV2 vectors were lacZ-positive, whereas incells over-expressing wtTC-PTP, or following pre-treatment with Tyr23,there was ˜13-fold and ˜20-fold increase, respectively, in transductionefficiency of ssAAV vectors (FIG. 4B). Treatment with MG132 for 4 hr (2hr for pretreatment and 2 hr for treatment together with AAV2 infection)led to ˜6-fold increase in transduction efficiency of ssAAV2 vectors(FIG. 4B). Surprisingly, however, the transduction efficiency of ssAAV2vectors following pretreatment with Tyr23, or TC-PTP over-expression,was not further enhanced by MG132. Similar results were obtained whenrAAV-EGFP vectors were used under identical conditions. As can be seenin FIG. 5A, whereas mock-infected cells showed no green fluorescence,and ˜15% of mock-treated cells transduced with scAAV2 vectors wereEGFP-positive, over-expression of TC-PTP, or pre-treatment with Tyr23led to ˜5-fold and ˜9-fold increase, respectively, in transductionefficiency of scAAV2 vectors (FIG. 5B). Treatment with MG132 led to˜5-fold increase in scAAV2 transduction efficiency (FIG. 5B). Thisincrease was not observed when the mTC-PTP expression plasmid was used.

It is noteworthy that the transduction efficiency of scAAV2 vectorsfollowing pre-treatment with Tyr23, or over-expression of TC-PTP, wasnot further enhanced by MG132 (FIG. 5B). Similar results were obtainedwhen lower viral particles/cell (1,000 and 2,000) ratios were used (FIG.9). These data further suggest that inhibition of EGFR-PTK signalingmodulates the ubiquitin/proteasome pathway, which affects aspects ofintracellular trafficking as well as second-strand DNA synthesis of AAV2vectors.

Inhibition of EGFR-PTK signaling decreases ubiquitination of AAV2 capsidproteins as well as total cellular proteins. The ubiquitin-proteasomepathway plays an important role in the cell by specifically degradingboth endogenous and foreign proteins.⁴⁷ A previous study⁴⁸ reported thatimmunoprecipitated AAV2 capsid proteins from infected cell lysates areconjugated with ubiquitin (Ub) and heat-denatured virus particles aresubstrates for in vitro ubiquitination. A more recently study⁴²documented that casein kinase II-induced phosphorylation of serineresidue 301 promotes ubiquitination and degradation of the bovinepapillomavirus E2 protein by the proteasome pathway. To further examinewhether EGFR-signaling is involved in ubiquitination of AAV2 capsidproteins, the following two sets of studies were performed: In the firststudy, cells were either mock-treated or treated with MG132, Tyr23, orboth, and cells either stably transfected with the wt- or mTC-PTPexpression plasmids were either mock-treated or treated with MG132 asdescribed supra. WCL were prepared and equivalent amounts of proteinswere subjected to Western blot analyses with anti-Ub monoclonalantibody. These results are shown in FIG. 6. Whereas the total level ofsmeary ubiquitinated cellular proteins was low in untreated cells (lanes1 and 6), and remained unchanged in Tyr23-treated cells (lane 3) as wellas in cells either stably transfected with wt- or mTC-PTP expressionplasmids (lanes 4 and 5), because these molecules are quickly degradedby the proteasome following ubiquitination, the significant accumulationof smeary ubiquitinated proteins in HeLa cells following inhibition ofproteasome activity by treatment with MG132 was observed as expected(lanes 2 and 7). Interestingly, however, Tyr23 treatment, orover-expression of wtTC-PTP, significantly decreased the accumulation ofMG132-induced ubiquitinated proteins (lanes 8 and 10), whereasover-expression of mTC-PTP had no effect (lane 9). In the second set,all mock-treated and treated cells were infected with AAV2 for 2 hrs at37° C. WCL were prepared at 4 hrs post-infection and equivalent amountsof proteins were immunoprecipitated first with anti-AAV2 capsid antibodyA20 followed by Western blot analyses with anti-Ub monoclonal antibody.

Similar results, shown in FIG. 7, indicate that whereas theubiquitinated AAV2 capsid proteins (Ub-AAV Cap, bracket) wereundetectable in mock-infected cells (lanes 1 and 2), the signal ofubiquitinated AAV2 capsid proteins was weaker in untreated cells (lane3), and remained unchanged in Tyr23-treated cells (lane 4) as well as incells stably transfected with wtTC-PTP expression plasmid (lane 7), asignificant accumulation of ubiquitinated AAV2 capsid proteins occurredfollowing treatment with MG132 (lane 5). However, treatment with Tyr23,or over-expression of wtTC-PTP dramatically inhibited the extent ofaccumulation of MG132-induced ubiquitinated AAV2 capsid proteins (lanes6 and 8). These results substantiate that inhibition of EGFR proteintyrosine kinase signaling also decreases ubiquitination of totalcellular proteins as well as AAV2 capsid proteins.

Discussion

In published studies' the inventors and their colleagues have documentedthat intracellular trafficking of AAV2 from cytoplasm to nucleus isimproved in murine hematopoietic stem cells from TC-PTP-transgenic mice.These data suggested that in addition to its crucial role in viralsecond-strand DNA synthesis, EGFR-PTK signaling was also involved inintracellular trafficking and/or nuclear transport of AAV2. Theubiquitin-proteasome pathway plays an essential role in AAV2intracellular trafficking, and proteasome inhibitors can promote AAV2nuclear transport, leading to augmentation of AAV2transduction.^(16, 31, 32) Direct evidence for ubiquitination of AAV2capsid proteins in HeLa cells and in in vitro ubiquitination assays hasbeen presented,⁴⁸ where only denatured AAV2 capsids, but not intactAAV2, could be ubiquitinated in vitro, which indicated that the intactAAV2 capsid required a conformational change or a modification, such asphosphorylation before its ubiquitination. A number of studies havereported that phosphorylation of cellular proteins by tyrosine orserine/threonine protein kinase is required for efficient ubiquitinationand degradation of these proteins.³⁶⁻⁴² For example, phosphorylation ofinhibitory κBα (IκBα) at serine residue #32 (Ser32) and serine residue#36 (Ser36) is a pre-requisite for cytokine-induced IκBα ubiquitinationand degradation.^(36, 37)

Receptor-mediated tyrosine kinase activation has been shown to be arequirement for T cell antigen receptor ubiquitination,³⁸ andubiquitination of CD16 ζ chain in human NK cells following receptorengagement has been shown to be tyrosine kinase-dependent.³⁹Modification of bovine papillomavirus E2 transactivator protein byubiquitination was reduced by mutation of serine residue #301 (Ser301),which indicated that phosphorylation of this residue was required forefficient ubiquitination and degradation of this protein by theubiquitin-proteasome pathway.⁴¹ Furthermore, casein kinase II-inducedphosphorylation of Ser301 in E2 protein induced a conformational changeand decreased the local thermodynamic stability of this region,promoting ubiquitination and targeted degradation of the E2 protein bythe proteasome pathway.⁴²

The present studies have demonstrated that EGFR-PTK signaling is indeedinvolved in the ubiquitin/proteasome pathway for modulation of nucleartransport of AAV2 vectors in addition to regulating viral second-strandDNA synthesis in HeLa cells. Similar results were also obtained with themurine fibroblast cell line NIH3T3, adult mouse hepatocyte cell lineH2.35, and fetal mouse hepatocyte cell line FL83B. Based on theavailable data, a model (shown schematically in FIG. 8) has beenpostulated, which helps explain the interactions between EGFR-PTKsignaling and ubiquitin/proteasome pathway in modulating intracellulartrafficking of AAV2 vectors as well as viral second-strand DNAsynthesis. In this model, following infection via binding to its primarycellular receptor, heparan sulfate proteoglycan (HSPG), and entrymediated by a co-receptor(s), such as FGFR1, AAV2 enters into the earlyendosome (EE) through clathrin-coated pits (CP)-mediated endocytosis.The EE then matures into late endosome (LE), in which AAV is degraded bylysosomal enzymes, if it fails to escape from the LE. If AAV2 escapesinto cytoplasm perinuclearly, it is ubiquitinated. It is hypothesizedthat EGFR-PTK-mediated phosphorylation of capsid proteins at tyrosineresidues is a prerequisite for ubiquitination.

A substantial number of ubiquitinated virions are then recognized anddegraded by cytoplasmic proteasomes on their way to the nucleus, leadingto inefficient nuclear transport (open arrow). In the presence ofproteasome inhibitors, vector degradation is reduced, leading to moreefficient nuclear transport of AAV. Inhibition of AAV2 capsidphosphorylation at tyrosine residues by EGFR-PTK inhibitors results indecreased ubiquitination of intact virions, which in turn, escapeproteasome-mediated degradation, an effect similar to what is seen withproteasome inhibitors. The net result is that intact virions enter thenucleus more efficiently (closed arrow). Following uncoating in thenucleus, the D-sequence in the AAV2 ITR forms a complex with FKBP52 [F],which is phosphorylated at tyrosine residues [P] by EGFR-PTK, andinhibits viral second-strand DNA synthesis. EGFR-PTK inhibitors preventphosphorylation of FKBP52 at tyrosine residues, and dephosphorylatedFKBP52 no longer binds to the AAV2 D-sequence, which in turn,facilitates viral second-strand DNA synthesis and efficient transgeneexpression ensues.

Consistent with this model, it was observed that AAV2 capsids can indeedbe phosphorylated at tyrosine residues by EGFR-PTK in in vitrophosphorylation assays, and that phosphorylated AAV2 virions transducecells much less efficiently.

Example 2—AAV2-Mediated Gene Transfer: Tyrosine Phosphorylation ofCapsid Proteins and its Consequences on Transgene Expression

The transduction efficiency of recombinant adeno-associated virus 2(AAV) vectors varies greatly in different cells and tissues in vitro andin vivo. Data from exemplary studies are illustrated in FIG. 11, FIG.12, FIG. 13, FIG. 14, and FIG. 15. Systematic studies were performed toelucidate the fundamental steps in the life cycle of AAV. For example,the inventors have shown that a cellular protein, FKBP52, phosphorylatedat tyrosine residues by epidermal growth factor receptor proteintyrosine kinase (EGFR-PTK), inhibits AAV second-strand DNA synthesis andconsequently, transgene expression in vitro^(24, 25) as well as invivo.^(19, 27, 28)

The inventors have also demonstrated that EGFR-PTK signaling modulatesthe ubiquitin/proteasome pathway-mediated intracellular trafficking aswell as FKBP52-mediated second-strand DNA synthesis of AAV vectors. Inthose studies, inhibition of EGFR-PTK signaling led to decreasedubiquitination of AAV capsid proteins, which in turn, facilitatednuclear transport by limiting proteasome-mediated degradation of AAVvectors, implicating EGFR-PTK-mediated phosphorylation of tyrosineresidues on AAV capsids.

The present example shows that AAV capsids can indeed be phosphorylatedat tyrosine residues by EGFR-PTK in in vitro phosphorylation assays, andthat phosphorylated AAV capsids retained their structural integrity.However, although phosphorylated AAV vectors could enter cells asefficiently as their unphosphorylated counterparts, their transductionefficiency was reduced. This reduction was not due to impaired viralsecond-strand DNA synthesis since transduction efficiency of bothsingle-stranded AAV (ssAAV) and self-complementary AAV (rAAV) vectorswas decreased by ˜68% and ˜74%, respectively. Intracellular traffickingof tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus wasalso significantly decreased, which most likely led to ubiquitination ofAAV capsids followed by proteasome-mediated degradation.

AAV capsids can be phosphorylated at tyrosine residues by EGFR-PTK in invitro phosphorylation assay and that phosphorylated AAV capsids retainedtheir structural integrity. Although phosphorylated AAV vectors couldenter cells as efficiently as their unphosphorylated counterparts, theirtransduction efficiency was significantly reduced. This reduction wasnot due to impaired viral second-strand DNA synthesis since transductionefficiency of both single-stranded AAV (ssAAV) and self-complementaryAAV (rAAV) vectors was decreased by ˜68% and ˜74%, respectively.Intracellular trafficking of tyrosine-phosphorylated AAV vectors fromcytoplasm to nucleus was also significantly decreased, most likely ledto ubiquitination of AAV capsids followed by proteasome-mediateddegradation. Taken together, these data illustrate that the complexinteractions occurring between EGFR-PTK signaling andubiquitin/proteasome pathway affects various aspects of intracellulartrafficking as well as second-strand DNA synthesis of AAV vectors.

Example 3—Next Generation rAAV2 Vectors: Point Mutations in TyrosinesLead to High-Efficiency Transduction at Lower Doses

The present example demonstrates that mutations of surface-exposedtyrosine residues on AAV2 capsids circumvents the ubiquitination step,thereby avoiding proteasome-mediated degradation, and resulting inhigh-efficiency transduction by these vectors in human cells in vitroand murine hepatocytes in vivo, leading to the production of therapeuticlevels of human coagulation factor at reduced vector doses. Theincreased transduction efficiency observed for tyrosine-mutant vectorsis due to lack of ubiquitination, and improved intracellular traffickingto the nucleus. In addition to yielding insights into the role oftyrosine phosphorylation of AAV2 capsid in various steps in the lifecycle of AAV2, these studies have resulted in the development of novelAAV2 vectors that are capable of high-efficiency transduction at lowerdoses.

Materials and Methods

Recombinant AAV2 vectors. Highly purified stocks of or scAAV2 vectorscontaining the enhanced green fluorescence protein (EGFP) gene driven bythe chicken β-actin (CBA) promoter (scAAV2-EGFP), and ssAAV2 vectorscontaining the factor IX (F.IX) gene under the control of theapolipoprotein enhancer/human α-1 antitrypsin (ApoE/hAAT) promoter(ssAAV2-F.IX) were generated as described previously.

Localization of surface-tyrosines on the AAV2 capsid surface. Thecrystal structure of AAV2 (PDB accession number 1lp3) was used tolocalize the tyrosine residues on the AAV2 capsid surface. Theicosahedral two-, three- and five-fold related VP3 monomers weregenerated by applying icosahedral symmetry operators to a referencemonomer using Program O on a Silicon graphics Octane workstation. Theposition of the tyrosine residues were then visualized and analyzed inthe context of a viral asymmetric unit using the program COOT, andgraphically presented using the program PyMOL Molecular Graphics System(DeLano Scientific, San Carlos, Calif., USA).

Construction of surface-exposed tyrosine residue mutant AAV2 capsidplasmid. A two-stage procedure, based on QuikChange II® site-directedmutagenesis (Stratagene, La Jolla, Calif.) was performed using plasmidpACG-2. Briefly, in stage one, two PCR extension reactions wereperformed in separate tubes for each mutant. One tube contained theforward PCR primer and the other contained the reverse primer (Table 2).In stage two, the two reactions were mixed and a standard PCRmutagenesis assay was carried out as per the manufacturer'sinstructions. PCR primers were designed to introduce changes fromtyrosine to phenylalanine residues as well as a silent change to createa new restriction endonuclease site for screening purposes (Table 2).All mutants were screened with the appropriate restriction enzyme andwere sequenced prior to use.

TABLE 2 NUCLEOTIDE SEQUENCES OF PRIMERS USEDFOR SITE-DIRECTED MUTAGENESIS Mutant SEQ ID NO:Primer Sequences (5′ to 3′) Y252F SEQ ID NO:1 CCCTGCCCACCTTCAACAACCACCTGTACAAACAAATTTCCAGCC                     Tyr-Phe  BsrGI Y272FSEQ ID NO:2 CCAATCAGGAGC TTCGAACGACAATCACTTCTTTGGCTACAG           BstBI         Tyr-Phe Y444F SEQ ID NO:3 CGACCAGTACCTGTATTTCTTAAGCAGAACAAACACTCCAAG             Tyr-Phe        Affil Y500FSEQ ID NO:4 CAACAACAGTGAATTCTCGTGG ACCGGTGCTACCAAGTACC            Tyr-Phe       Agel Y700F SEQ ID NO:5GGAATCCCGAAATTCAGTTCACTTCGAACTACAACAAGTCTG            Tyr-Phe       BstBI Y704F SEQ ID NO:6GGAATCCCGAAATTCAGTACACTTCGAACTTCAACAAGTCTG           BstBI          Tyr-Phe Y730F SEQ ID NO:7 CCTCGCCCCATTGGTACCAGATTCCTGACTCGTAATC          Acc65I        Tyr-Phe

Preparation of whole cell lysates (WCL) and co-immunoprecipitations. WCLwere prepared as described. Approximately 2×10⁶ HeLa cells, mock-treatedor treated with MG132, were also subjected to mock-infection orinfection with the WT scAAV2-EGFP or Y730F mutant vectors at 5×10³particles/cell for 2 hr at 37° C. For immunoprecipitations, cells weretreated with 0.01% trypsin and washed extensively with PBS. WCL werecleared of non-specific binding by incubation with 0.25 mg of normalmouse IgG together with 20 μl of protein G-agarose beads. Afterpreclearing, 2 μg of capsid antibody against intact AAV2 particles(mouse monoclonal IgG₃, clone A20; Research Diagnostics, Inc. (Flanders,N.J.), or 2 μg of normal mouse IgG (as a negative control) were addedand incubated at 4° C. for 1 hr, followed by precipitation with proteinG-agarose beads. For immunoprecipitations, resuspended pellet solutionswere used for SDS-PAGE. Membranes were treated with monoclonalHRP-conjugated anti-Ub antibody (1:2,000 dilution) specific forubiquitin (Ub) (mouse monoclonal immunoglobulin G₁ □IgG₁], clone P4D1;Santa Cruz, Calif.) Immunoreactive bands were visualized usingchemiluminescence (ECL-plus, Amersham Pharmacia Biotech, Piscataway,N.J.).

Isolation of nuclear and cytoplasmic fractions from HeLa cells. Nuclearand cytoplasmic fractions from HeLa cells were isolated andmock-infected or recombinant wt scAAV2-EGFP or Y700F vector-infectedcells were used to isolate the cytoplasmic and nuclear fractions. Thepurity of each fraction was determined to be >95%.

Southern blot analysis for AAV2 trafficking. Low-M_(r) DNA samples fromnuclear and cytoplasmic fractions were isolated and electrophoresed on1% agarose gels or 1% alkaline-agarose gels followed by Southern blothybridization using a ³²P-labeled EGFP-specific DNA probe.

Recombinant AAV2 vector transduction assays in vitro. Approximately1×10⁵ HeLa cells were used for transductions with recombinant AAV2vectors. The transduction efficiency was measured 48 hrpost-transduction by EGFP imaging using fluorescence microscopy. Imagesfrom three to five visual fields were analyzed quantitatively by ImageJanalysis software (NIH, Bethesda, Md., USA). Transgene expression wasassessed as total area of green fluorescence (pixel²) per visual field(mean±SD). Analysis of variance (ANOVA) was used to compare between testresults and the control and they were determined to be statisticallysignificant.

Recombinant AAV2 vector transduction studies in vivo. scAAV2-EGFPvectors were injected intravenously via the tail vein into C57BL/6 miceat 1×10¹⁰ virus particles per animal. Liver sections from three hepaticlobes of the mock-injected and injected mice 2 weeks after injectionwere mounted on slides. The transduction efficiency was measured by EGFPimaging as described. ssAAV2-FI.X vectors were injected intravenously(via the tail vein) or into the portal vein of C57BL/6, BALB/c, andC3H/HeJ mice at 1×10¹⁰ or 1×10¹¹ virus particles per animal. Plasmasamples were obtained by retro-orbital bleed and analyzed for hF.IXexpression by ELISA.

Results

Mutations in surface-exposed tyrosine residues significantly improve thetransduction efficiency of AAV2 vectors in HeLa cells in vitro. Todemonstrate that tyrosine-phosphorylation of AAV2 capsids leads toincreased ubiquitination and results in impaired intracellulartrafficking, and is therefore unfavorable to viral transduction,surface-exposed tyrosine residues were modified on AAV2 capsids.Inspection of the capsid surface of the AAV2 structure revealed a totalof 7 surface-exposed tyrosine residues (Y252, Y272, Y444, Y500, Y700,Y704, and Y730). Site-directed mutagenesis was performed for each of the7 tyrosine residues, which were conservatively substituted withphenylalanine residues (tyrosine-phenylalanine, Y-F) (Table 2).scAAV2-EGFP genomes encapsidated in each of the tyrosine-mutant capsidswere successfully packaged (Table 3), and mutations of thesurface-exposed tyrosine residues did not lead to reduced vectorstability.

TABLE 3 TITERS OF WILDTYPE (WT) AND TYROSINE- MODIFIED (Y-F MUTANTS)AAV2 VECTORS 1^(st) packaging 2^(nd) packaging 3^(rd) packaging 4^(th)packaging AAV Vectors titers (vgs/ml) titers (vgs/ml) titers (vgs/ml)titers (vgs/ml) WT scAAV2-EGFP 3.4 × 10¹¹ 1.0 × 10¹² 3.2 × 10¹¹ 3.0 ×10¹¹ Y252F scAAV2-EGFP 3.8 × 10¹¹ 4.0 × 10¹¹ ND ND Y272 scAAV2-EGFP 7.7× 10¹¹ 1.0 × 10¹¹ ND ND Y444F scAAV2-EGFP 9.7 × 10¹⁰ 4.0 × 10¹⁰ 6.0 ×10⁹  5.0 × 10¹⁰ Y500F scAAV2-EGFP 8.8 × 10¹⁰ 2.0 × 10⁹  4.0 × 10¹⁰ 6.0 ×10¹⁰ Y700F scAAV2-EGFP 1.0 × 10¹¹ 4.0 × 10¹¹ ND ND Y704F scAAV2-EGFP 6.0× 10¹¹ 2.0 × 10¹¹ ND ND Y730F scAAV2-EGFP 1.2 × 10¹¹ 5.0 × 10¹¹ 1.2 ×10¹¹ 4.0 × 10¹¹ ND = Not done.

The transduction efficiency of each of the tyrosine-mutant vectors wasanalyzed and compared with the WT scAAV2-EGFP vector in HeLa cells invitro under identical conditions. From the results it was evident thatwhereas mock-infected cells showed no green fluorescence, thetransduction efficiency of each of the tyrosine-mutant vectors wassignificantly higher compared with the WT scAAV2-EGFP vector at 2,000viral particles/cell. Specifically, the transduction efficiency ofY444F, Y500F, Y730F vectors was ˜8- to 11-fold higher than the WTvector.

Mutations in surface-exposed tyrosine residues dramatically improve thetransduction efficiency of AAV2 vectors in murine hepatocytes in vivo.The efficacy of WT and tyrosine-mutant scAAV2-EGFP vectors was alsoevaluated in a mouse model in vivo. As can be seen in FIG. 17A, thetransduction efficiency of tyrosine-mutant vectors was significantlyhigher, and ranged between 4-29-fold, compared with the WT vector (FIG.17B). When other tissues, such as heart, lung, kidney, spleen, pancreas,GI tract (jejunum, colon), testis, skeletal muscle, and brain wereharvested from mice injected with 1×10¹⁰ particles of thetyrosine-mutant vectors and analyzed, no evidence of EGFP geneexpression was seen. Thus, mutations in the surface-exposed tyrosineresidues did not appear to alter the liver-tropism following tail veininjection of these vectors in vivo.

The increased transduction efficiency of tyrosine-mutant vectors is dueto lack of ubiquitination, and improved intracellular trafficking to thenucleus. To further confirm the hypothesis that EGFR-PTK-mediatedphosphorylation of capsid proteins at tyrosine residues is apre-requisite for ubiquitination of AAV2 capsids, and that ubiquitinatedvirions are recognized and degraded by cytoplasmic proteasome on theirway to the nucleus, leading to inefficient nuclear transport, a seriesof experiments were performed as follows.

In the first set of studies, HeLa C12 cells, carryingadenovirus-inducible AAV2 rep and cap genes, were mock-infected orinfected with WT, Y444F or Y730F scAAV2-EGFP vectors. As shown in FIG.18A and FIG. 18B, whereas mock-infected cells showed no greenfluorescence, and ˜15% of cells were transduced with the WT scAAV2-EGFPvectors in the absence of co-infection with adenovirus, the transductionefficiency of Y444F and Y730F scAAV2-EGFP vectors was increased by ˜9and ˜18-fold, respectively, compared with the WT vector. Interestingly,whereas co-infection with adenovirus led to ˜11-fold increase (cf. FIG.18B), the transduction efficiency of Y444F and Y730F scAAV2-EGFP vectorswas not further enhanced by co-infection with adenovirus. Sinceadenovirus can improve AAV2 vector nuclear transport in HeLa cells,these data suggest that the surface-exposed tyrosine residues play arole in intracellular trafficking of AAV2, and that their removal leadsto efficient nuclear transport of AAV2 vectors.

In the second set of studies, HeLa cells, either mock-treated or treatedwith Tyr23, a specific inhibitor of EGFR-PTK, or MG132, a proteasomeinhibitor, both known to increase the transduction efficiency of AAVvectors, were mock-infected or infected with the WT or Y730F scAAV2-EGFPvectors. These data are shown in FIG. 18C. Whereas mock-infected cellsshowed no green fluorescence, and ˜5% of cells were transduced with theWT scAAV2-EGFP vectors in mock-treated cells, pretreatment with Tyr23 orMG132 led to an ˜9-fold and ˜6-fold increase in the transductionefficiency, respectively (FIG. 18D). Although the transductionefficiency of Y730F scAAV2-EGFP vectors was increased by ˜14-foldcompared with the WT vectors, it was not further enhanced bypretreatment with either Tyr23 or MG132 (FIG. 18D). These data stronglysuggest that the absence of surface-exposed tyrosine residues, whichprevented phosphorylation of the mutant vectors, likely preventedubiquitination of the capsid proteins, and these vectors on their way tothe nucleus could not be recognized and degraded by the proteasome,which led to their efficient nuclear translocation.

In the third set of studies, HeLa cells, either mock-treated or treatedwith MG132, were mock-infected or infected with the WT, Y730F, or Y444FscAAV2-EGFP vectors. WCL were prepared 4 hrs post-infection andequivalent amounts of proteins were immunoprecipitated first withanti-AAV2 capsid antibody (A20) followed by Western blot analyses withanti-Ub monoclonal antibody. These results are shown in FIG. 19. As canbe seen, whereas ubiquitinated AAV2 capsid proteins (Ub-AAV2 Cap) wereundetectable in mock-infected cells (lanes 1, 2), the signal ofubiquitinated AAV2 capsid proteins was weaker in untreated cells (lanes3, 5), and a significant accumulation of ubiquitinated AAV2 capsidproteins occurred following treatment with MG132 (lane 4).Interestingly, infections with Y730F or Y444F vectors dramaticallydecreased the extent of accumulation of MG132-induced ubiquitinated AAV2capsid proteins (lanes 6, 8). These results substantiate that mutationin tyrosine residues circumvents proteasome-mediated degradation of thevectors.

In the fourth set of studies, the fate of the input WT, Y444F, and Y730Fvector viral DNA was determined in HeLa cells. Southern blot analysis oflow-M_(r) DNA samples isolated from cytoplasmic [C] and nuclear [N]fractions (FIG. 5A) and densitometric scanning of autoradiographs (FIG.20B), revealed that ˜36% of the input scAAV2 DNA was present in thenuclear fraction in cells infected with the WT vector (FIG. 20A, lane 4and FIG. 20B), consistent with previous studies. Interestingly, however,the amount of input Y730F and Y444F scAAV2 vector DNA in the nuclearfraction was increased to ˜72% and ˜70%, respectively (FIG. 20B). Theseresults further document that mutations in the surface-exposed tyrosineresidues prevent ubiquitination of AAV2 capsids, resulting in a decreaseof proteasome-mediated degradation, and in turn, facilitate nucleartransport of AAV2 vectors.

Tyrosine-mutant vectors express therapeutic levels of human Factor IXprotein at ˜10-fold reduced vector dose in mice. It was important toexamine whether tyrosine-mutant AAV2 vectors were capable of deliveringa therapeutic gene efficiently at a reduced vector dose in vivo. To thisend, a single-stranded, hepatocyte-specific human Factor IX (h.FIX)expression cassette was encapsidated in the Y730F vector, and theefficacy of this vector was tested in 3 different strains of mice(BALB/c, C3H/HeJ, and C57BL/6). Consistently in all 3 strains, Y730Fvector achieved ˜10-fold higher circulating hF.IX levels compared withthe WT vector following tail vein or portal vein administration, withthe latter being the more effective route. These results, shown in FIG.21A, FIG. 21B, FIG. 21C, and FIG. 21D, clearly indicate that the Y730Fvectors expressed therapeutic levels of human F.IX protein (˜50 ng/ml)at ˜10-fold reduced vector dose (10¹⁰ particles/mouse) in C57BL/6 miceby port vein injection. It should be noted that hepatic viral genetransfer in C57BL/6 mice is generally more efficient than in the othertwo strains.

These results demonstrated here are consistent with the interpretationthat EGFR-PTK-induced tyrosine phosphorylation of AAV2 capsid proteinspromotes ubiquitination and degradation of AAV2, thus leading toimpairment of viral nuclear transport and decrease in transductionefficiency. Mutational analyses of each of the seven surface-exposedtyrosine residues yield AAV2 vectors with significantly increasedtransduction efficiency in vitro as well as in vivo. Specifically, Y444Fand Y730F mutant vectors bypass the ubiquitination step, which resultsin a significantly improved intracellular trafficking and delivery ofthe viral genome to the nucleus.

Despite long-term therapeutic expression achieved in preclinical animalmodels by AAV2 vectors composed of the WT capsid proteins, in a recentgene therapy trial, two patients with severe hemophilia B developedvector dose-dependent transaminitis that limited duration ofhepatocyte-derived hF.IX expression to <8 weeks. Subsequent analysesdemonstrated presence of memory CD8⁺ T cells to AAV capsids in humansand an MHC I-restricted, capsid-specific cytotoxic T lymphocyte (CTL)response in one of the hemophilia B patients, which mirrored the timecourse of the transaminitis. It was concluded that this CD8⁺ T cellresponse to input capsid eliminated AAV2-transduced hepatocytes. Thepresent studies show that a lower capsid antigen dose is sufficient forefficient gene transfer with the Y730F vector. The data also showmuch-reduced ubiquitination of AAV-Y730F compared to WT capsid, aprerequisite for MHC I presentation. Thus, the T-cell response to AAV2capsid (a serious hurdle for therapeutic gene transfer in the liver),may be avoided by using the surface-exposed tyrosine-mutant AAV2vectors.

Dramatically increased transduction efficiency of tyrosine-mutantvectors have also been observed in primary human neuronal andhematopoietic stem cells in vitro and in various tissues and organs inmice in vivo. Double, triple, and quadruple tyrosine-mutants have alsobeen constructed to examine whether such multiple mutants are viable,and whether the transduction efficiency of these vectors can beaugmented further. It is noteworthy that with a few exceptions (Y444positioned equivalent to a glycine in AAV4 and arginine in AAV5; Y700positioned equivalent to phenylalanine in AAV4 and AAV5; and Y704positioned equivalent to a phenylalanine in AAV7), these tyrosineresidues are highly conserved in AAV serotypes 1 through 10.

Example 4—Analysis of Tyrosine Positions on the AAV2 Capsid

The following summary is a list of all Tyr residues in the structurallyordered region of VP3 (217-735):

These Tyr residues are classified based upon whether they are exposed,partially hidden, or not exposed:

Surface Exposed

Tyr252¹— Surface exposed—canyon floor

Tyr272—Surface exposed—raised region between the 2- and 5-folddepressions

Tyr444—Surface exposed—wall of 3-fold protrusions

Tyr500—Surface exposed—wall of 3-fold protrusions

Tyr700—Surface exposed—2-fold axis

Tyr704²—Surface exposed—2-fold axis

Tyr730—Surface exposed—2-fold axis

¹Tyr252 has been mutated and confirmed by partial sequencing. ²Tyr704has been muted and completely sequenced. All others listed have alsobeen mutated, with sequence analysis being performed to confirm each.

Surface, but Mostly Hidden Tyrosine Residues

Tyrosine resides Tyr275, Tyr281, Tyr508, Tyr576, Tyr612, Tyr673, andTyr720.

Not Exposed

Tyr257, Tyr348, Tyr352, Tyr375, Tyr377, Tyr393, Tyr397, Tyr413, Tyr424,Tyr441, Tyr443, and Tyr483.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1-36. (canceled)
 37. A modified adeno associated virus (AAV) capsidprotein comprising a non-native amino acid at one or more positions thatcorrespond to Tyr576 or Tyr700 of a wild-type AAV2 capsid protein. 38.The modified AAV capsid protein according to claim 37, wherein thenon-native amino acid is a phenylalanine.
 39. The modified AAV capsidprotein according to claim 37, wherein the capsid protein is an AAVserotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5 or AAV6 capsid protein. 40.The modified AAV capsid protein according to claim 37, wherein thecapsid protein is comprised within an AAV particle.
 41. A recombinantadeno-associated viral (rAAV) particle that comprises the modified AAVcapsid protein according to claim
 37. 42. The rAAV particle of claim 41,wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6particle.
 43. The rAAV particle of claim 41, wherein the transductionefficiency of the rAAV particle comprising the modified AAV capsidprotein is higher than that of an AAV particle comprising acorresponding, unmodified AAV capsid protein.
 44. The rAAV particleaccording to claim 41, further comprising a nucleic acid segment thatencodes a therapeutic agent or a diagnostic agent operably linked to apromoter capable of expressing the nucleic acid segment in a host cell.45. The rAAV particle according to claim 44, further comprising anenhancer sequence operably linked to the nucleic acid segment.
 46. TherAAV particle according to claim 45, wherein the enhancer sequence is aCMV enhancer, a synthetic enhancer, a liver-specific enhancer, avascular-specific enhancer, a brain-specific enhancer, a neuralcell-specific enhancer, a lung-specific enhancer, a muscle-specificenhancer, a kidney-specific enhancer, a pancreas-specific enhancer, oran islet cell-specific enhancer.
 47. The rAAV particle according toclaim 44, wherein the promoter is a heterologous, tissue-specific,constitutive or inducible promoter.
 48. The rAAV particle according toclaim 47, wherein the promoter is a CMV promoter, a β-actin promoter, aninsulin promoter, an enolase promoter, a BDNF promoter, an NGF promoter,an EGF promoter, a growth factor promoter, an axon-specific promoter, adendrite-specific promoter, a brain-specific promoter, ahippocampal-specific promoter, a kidney-specific promoter, an elafinpromoter, a cytokine promoter, an interferon promoter, a growth factorpromoter, an alpha-1 antitrypsin promoter, a brain-specific promoter, aneural cell-specific promoter, a central nervous system cell-specificpromoter, a peripheral nervous system cell-specific promoter, aninterleukin promoter, a serpin promoter, a hybrid CMV promoter, a hybridβ-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter, aTet-inducible promoter, or a VP16-LexA promoter.
 49. The rAAV particleaccording to claim 44, wherein the therapeutic agent is a polypeptide, apeptide, an antibody, an antigen binding fragment, a ribozyme, a peptidenucleic acid, an siRNA, an RNAi, an antisense oligonucleotide, or anantisense polynucleotide.
 50. A composition comprising the rAAV particleaccording to claim
 41. 51. A composition comprising the rAAV particleaccording to claim
 44. 52. A method for administering a therapeutic or adiagnostic agent to a mammal in need thereof, the method comprising:providing to a cell, tissue or organ of the mammal the compositionaccording to claim 51, wherein the transduction efficiency of the rAAVparticle comprised in the composition is higher than that of an AAVparticle comprising a corresponding, unmodified, wild-type capsidprotein.
 53. The method of claim 52, wherein the rAAV particle is anAAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 particle.
 54. The method accordingto claim 52, wherein the transduction efficiency of the rAAV particlecomprising the modified capsid protein is at least 4-fold higher thanthat of an AAV particle comprising the corresponding, unmodified capsidprotein.
 55. The method according to claim 52, wherein the non-nativeamino acid is a phenylalanine.
 56. The method according to claim 52,wherein the rAAV particle is comprised within a mammalian host cell. 57.The method according to claim 56, wherein the mammalian host cell is ahuman endothelial, epithelial, vascular, liver, lung, heart, pancreas,intestinal, kidney, muscle, bone, neural, blood, or brain cell.
 58. Amethod of providing a therapeutic peptide, polypeptide, or RNA to amammal in need thereof, the method comprising: introducing into aselected population of cells of the mammal an effective amount of arecombinant adeno-associated viral (rAAV) particle according to claim41; wherein the rAAV particle comprises a nucleic acid segment thatencodes the therapeutic peptide, polypeptide, or RNA, and that isoperably linked to at least one promoter that expresses the nucleic acidsegment in one or more cells of the selected population.
 59. The methodaccording to claim 58, wherein the rAAV particle is an AAV1, AAV2, AAV3,AAV4, AAV5 or AAV6 particle.
 60. The method according to claim 58,wherein the therapeutic peptide or polypeptide is an antibody or anantigen-binding fragment thereof; or wherein the therapeutic RNA is aribozyme, a peptide-nucleic acid, an siRNA, an RNAi, or an antisenseoligonucleotide; and/or wherein the promoter is a heterologous promoter,a tissue-specific promoter, a constitutive promoter, a cell-specificpromoter, an inducible promoter, or any combination thereof.
 61. Themethod according to claim 58, wherein the nucleic acid segment isfurther operably linked to at least one enhancer sequence.
 62. Themethod according to claim 58, wherein the selected population of cellsis a population of human endothelial, epithelial, vascular, liver, lung,heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, orbrain cells.
 63. The method according to claim 58, wherein thetherapeutic peptide, polypeptide, or RNA, is expressed in one or morecells of the selected population in an amount, and for a time sufficientto treat or ameliorate at least one symptom of a disease, a disorder, adysfunction, an injury, or trauma in a human patient.
 64. The methodaccording to claim 58, wherein the mammal has cancer, diabetes,autoimmune disease, kidney disease, cardiovascular disease, pancreaticdisease, intestinal disease, liver disease, neurological disease,neuromuscular disease, Batten disease, Alzheimer's disease, Huntingtondisease, Parkinson's disease, pulmonary disease, an α-1 antitrypsindeficiency, a neurological disability, a neuromotor deficit, aneuroskeletal impairment, ischemia, stroke, or any combination thereof.65. A method of treating or ameliorating one or more symptoms of adisease, a disorder, a dysfunction, an injury, or trauma in a mammal,the method comprising administering to the mammal an effective amount ofthe recombinant adeno-associated viral (rAAV) particle according toclaim 41; wherein the particle comprises a nucleic acid segment thatencodes a therapeutic peptide, polypeptide, or RNA, and that is operablylinked to at least one promoter that expresses the nucleic acid segmentin one or more cells of a selected population.
 66. The method of claim65, wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6particle.
 67. The method according to claim 65, wherein the one or morecells are human endothelial, epithelial, vascular, liver, lung, heart,pancreas, intestinal, kidney, muscle, bone, neural, blood, or braincells.